bluuebunny/crossref_metadata_2025 huggingface dataset:
Rational design of nonnatural peptides as high-affinity ligands for the HLA-B*2705 human leukocyte antigen.
Authors: Rognan D , Scapozza L , Folkers G , Daser A
Published: May 2006

From the three-dimensional structure of the class I major histocompatibility complex (MHC) HLA-B*2705 protein, several nonnatural peptides were designed either to optimize the interactions of one peptide amino acid (position 3) with its HLA binding pocket (pocket D) or to simplify the T-cell receptor-binding part by substitution with organic spacers. The stability of each MHC-ligand complex was simulated by 150-ps molecular dynamics in a water environment and compared with that of the natural complexes. All peptides were synthesized and tested for binding to the class I MHC protein in an in vitro assembly assay. As predicted from the computed atomic fluctuations and buried surface areas of MHC-bound ligands, bulky hydrophobic side chains at position 3 enhance the binding of a nonameric peptide to the HLA-B27 protein. Furthermore, it was possible to simplify half of the peptide sequence (residues 4-8) by replacement with organic fragments without altering the affinity of the designed ligands for the class I MHC protein. This study constitutes an initial step toward the rational design of nonpeptide class I MHC ligands for use in the selective immunotherapy of autoimmune diseases associated with particular HLA alleles.
Passive immunotherapy in AIDS: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma.
Authors: Vittecoq D , Chevret S , Morand-Joubert L , Heshmati F , Audat F , Bary M , Dusautoir T , Bismuth A , Viard J P , Barré-Sinoussi F
Published: May 2006

A randomized double-blind controlled trial was conducted to determine the efficacy of passive immunotherapy in the treatment of symptomatic human immunodeficiency virus (HIV) infection. This trial included 86 symptomatic patients randomized to receive plasma rich in anti-HIV-1 antibody or standard seronegative plasma. Each patient in both groups received a 300-ml infusion every 14 days over a 1-year period, and every 28 days thereafter, in addition to zidovudine and other conventional prophylactic treatments. Plasma donors were selected among symptomless seropositive individuals with a CD4 lymphocyte count > or = 400 x 10(6) cells per liter, a negative p24 antigen assay, and a high concentration of anti-p24 antibody. The plasmas were heat-inactivated before infusion. During the study period (day 28-day 365) scheduled by the protocol, clinical benefit from passive immunotherapy was observed in delaying the appearance of the first AIDS-defining event (P < 0.009) and reducing the cumulative incidence of such events, which was estimated 3-fold higher in the control group compared to the treatment group. Seven deaths occurred in the treatment group vs. 11 in the control group (P = 0.27). A total of 47 patients died or exhibited new AIDS-defining events, 18 in the treatment group and 29 in the control group (P = 0.009). No clinical benefit was observed after the 1-year period with infusions performed every 4 weeks. These results indicate a favorable effect of passive immunotherapy on the evolution of advanced AIDS.
A single arginine residue determines species specificity of the human growth hormone receptor.
Authors: Souza S C , Frick G P , Wang X , Kopchick J J , Lobo R B , Goodman H M
Published: May 2006

Although growth hormone (GH) receptors (GHRs) in many species bind human (h) GH as well as their own GH, the hGHR only binds primate GH. Arg43 in hGHR interacts with Asp171 of hGH. Nonprimates have a His in the position equivalent to residue 171 of primate GH and a Leu in position 43 of primate GHR. To determine whether Arg43 accounts for the species specificity of the hGHR, point mutations that changed Leu43 to Arg were introduced into the cDNAs encoding the bovine (b) GHR or the rat GH binding protein (GHBP) and these mutants or their wild-type (WT) counterparts were expressed in mouse L cells. Binding of hGH or bGH to transfected cells or to GHBP secreted into the incubation medium was assessed by displacement of 125I-labeled hGH. WT and mutant bGHR bound hGH with similar affinity, but the affinity of the mutant receptors for bGH was reduced 200-fold. Likewise, WT and mutant GHBP bound hGH with equal affinity, but only WT GHBP bound bGH. Cross-linking of 125I-labeled hGH to WT or mutant GHR produced a 141-kDa labeled complex whose appearance was blocked by unlabeled hGH, but bGH blocked cross-linking only to WT receptors. Both hGH and bGH stimulated tyrosine phosphorylation of a 95-kDa protein in cells transfected with WT GHR, but bGH was less effective in cells expressing mutant GHR. We conclude that incompatibility of Arg43 in the hGHR with His171 in nonprimate GH is the major determinant of species specificity.
A diabetes-associated T-cell autoantigen maps to a telomeric locus on mouse chromosome 6.
Authors: Dallas-Pedretti A , McDuffie M , Haskins K
Published: May 2006

Identification of diabetes-associated T-cell autoantigens is important for understanding the immunopathology of diabetes and developing improved therapeutic strategies. We have used a genetic approach to move toward identifying the autoantigen recognized by a diabetogenic islet-specific T-cell clone from a nonobese diabetic (NOD) mouse. The unique antigen recognition pattern of this clone was utilized to map the gene encoding the antigen (or its expression) by genetic linkage analysis. In vitro analysis of T-cell proliferation by this clone showed that the capacity of the islets to stimulate T cells segregates as a single codominant gene in BALB/cByJ x (BALB/cByJ x NOD/Bdc) backcross mice. This phenotype was tightly linked to two microsatellites in the telomeric region of mouse chromosome 6.
Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages.
Authors: Winston B W , Lange-Carter C A , Gardner A M , Johnson G L , Riches D W
Published: May 2006

Tumor necrosis factor alpha (TNF alpha) is bound by two cell surface receptors, CD120a (p55) and CD120b (p75), that belong to the TNF/nerve growth factor receptor family and whose signaling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signaling events activated by receptor crosslinking are unknown, although activation of the mitogen-activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a. In this study, we investigated the upstream kinases that mediate the activation of the 42-kDa MAPK p42mapk/erk2 following crosslinking of CD120a in mouse macrophages. Exposure of mouse macrophages to TNF alpha stimulated a time-dependent increase in the activity of MAPK/ERK kinase (MEK) that temporally preceded peak activation of p42mapk/erk2. MEKs, dual-specificity threonine/tyrosine kinases, act as a convergence point for several signaling pathways including Ras/Raf, MEK kinase (MEKK), and Mos. Incubation of macrophages with TNF alpha was found to transiently stimulate a MEKK that peaked in activity within 30 sec of exposure and progressively declined toward basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNF alpha is mediated by the sequential activation of a MEKK and a MEK in a c-Raf-1- and Raf-B-independent fashion.
Molecular chaperones involved in protein degradation in the endoplasmic reticulum: quantitative interaction of the heat shock cognate protein BiP with partially folded immunoglobulin light chains that are degraded in the endoplasmic reticulum.
Authors: Knittler M R , Dirks S , Haas I G
Published: May 2006

In the absence of immunoglobulin heavy-chain expression, some immunoglobulin light (L) chains are retained and degraded within the cell. We investigated the fate of two different nonsecreted murine L chains which exhibit different half-lives (50 min and 3-4 hr). Our results demonstrate that both nonsecreted L chains are quantitatively bound to BiP as partially oxidized molecules. The kinetics of L-chain degradation coincided with those of L-chain dissociation from BiP, which suggests that these two processes are functionally related. L-chain degradation does not depend on vesicular transport, indicating that these soluble proteins are degraded in the endoplasmic reticulum (ER). In contrast, secreted L chains, which interact only transiently with BiP, are completely oxidized and are not degraded even when they are artificially retained in the ER. Our data support the model that, by means of BiP interaction, the ER degradation mechanism has the potential to discriminate between partially and completely folded molecules.
Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.
Authors: Boffa L C , Carpaneto E M , Allfrey V G
Published: May 2006

An amplification of tandem CAG trinucleotide sequences in DNA due to errors in DNA replication is involved in at least four hereditary neurodegenerative diseases. The CAG triplet repeats when translated into protein give rise to tracts of glutamine residues, which are a prominent feature of many transcription factors, including the TATA-binding protein of transcription factor TFIID. We have used a biotin-labeled, complementary peptide nucleic acid (PNA) to invade the CAG repeats in intact chromatin and then employed a method for the selective isolation of transcriptionally active chromatin restriction fragments containing the PNA.DNA hybrids. The PNA-containing chromatin fragments were captured on streptavidin-agarose magnetic beads and shown to contain all the CAG.PNA hybrids of the active chromatin fraction. DNA hybridization experiments using a DNA probe specific for unique sequences downstream of the CAG-tandem repeats confirmed that the PNA.DNA hybrids contained the transcribed gene for the TATA-binding protein. In contrast, no hybridization signal was detected with a DNA probe specific for the c-myc protooncogene, which is amplified and transcriptionally active in COLO 320DM cells but lacks CAG tandem repeats.
GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs.
Authors: Rodnina M V , Wintermeyer W
Published: May 2006

The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system. The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile ... (mMFTI) or Met-Phe-Phe-Gly ... (mMFFG) or with poly(U). The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography. With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess. Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used. In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed. In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP. Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated. The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.
Biosynthesis of a biologically active single peptide chain containing the human common alpha and chorionic gonadotropin beta subunits in tandem.
Authors: Sugahara T , Pixley M R , Minami S , Perlas E , Ben-Menahem D , Hsueh A J , Boime I
Published: May 2006

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common alpha subunit and a hormone-specific beta subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormone-specific posttranslational modifications, and signal transduction. To address whether alpha and beta subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) beta subunit genetically fused to the alpha subunit was constructed. The resulting polypeptide hCG molecule not only was efficiently secreted but also displayed an increased biological activity in vitro and in vivo. These data show that the alpha and hCG beta subunits encoded as a single chain retain a biologically active conformation similar to that seen in the heterodimer. This approach can be used to investigate structure-function relationships of the glycoprotein hormone family that were previously not tractable because of the absolute dependence on assembly for the biological response. Moreover, other bioactive multisubunit ligands can be engineered where the combination efficiency and specificity of heterodimers and homodimers are otherwise difficult to control.
Cisplatin inhibits chromatin remodeling, transcription factor binding, and transcription from the mouse mammary tumor virus promoter in vivo.
Authors: Mymryk J S , Zaniewski E , Archer T K
Published: May 2006

The anticancer drug cis-diamminedichloro-platinum(II) (cisplatin) covalently modifies DNA, and these lesions are thought to lead to cell death by inhibiting DNA and RNA synthesis. By using in vivo analysis techniques, we have investigated the influence of cisplatin on hormone-induced transcription from the mouse mammary tumor virus (MMTV) promoter. Cisplatin substantially reduced glucocorticoid-induced expression from the MMTV promoter stably incorporated into mouse tumor cells. The glucocorticoid-receptor-dependent chromatin remodeling and loading of transcription factors that is a signature response of this promoter in the context of chromatin were significantly reduced by cisplatin but not by the clinically ineffective trans-isomer trans-diamminedichloroplatinum(II) (transplatin). Additional in vivo studies on transiently introduced nonchromatin MMTV templates demonstrated that cisplatin modification of DNA blocked binding of the transcription factor NF1. These results provide strong evidence that cisplatin influences transcription by interfering with the opening of repressive chromatin structures and by blocking transcription factor binding directly, each of which could contribute substantially to its toxicity.
Cell membrane formation during the cellularization of the syncytial blastoderm of Drosophila.
Authors: Loncar D , Singer S J
Published: May 2006

The early blastoderm of Drosophila is a syncytium in which about 6000 nuclei become localized in the peripheral cytoplasm. During cycle 14 interphase, a wave of membrane formation encircles each nucleus inside its own plasma membrane, thereby generating an intact epithelial layer. The details of this process of cellularization have been unclear. Using an improved method of fixation of the embryos for electron microscopy, we show by morphological observations that a large number of membrane-bounded, electron-transparent vesicles, of diameters ranging from 0.05 micron to 0.5 micron, are present in the periplasm and become redistributed during cellularization so as to provide the membrane mass required at each phase of the process. We recognize three phases. In the first two phases, the vesicles that were present in the apical periplasmic space at earlier stages become concentrated and aligned between the nuclei. The vesicles then undergo concerted but not precisely synchronous fusion to form double membranes, starting at furrows in the plasma membrane of the embryo and extending about 7 microns into the periplasmic space. Subsequently, in the third phase vesicles are recruited to the basal periplasmic space but do not become aligned between the nuclei as in the first phase. We presume that these vesicles fuse individually with the growing ends of the double membranes until encirclement of each nucleus is complete. We speculate that these vesicles are all derived from the Golgi apparatus and are moved about in the blastoderm by interactions with components of the cytoskeleton.
A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase.
Authors: Sun H , Seyer J M , Patel T B
Published: May 2006

Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs. Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs. A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs. This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein. Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi. Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs. Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein. Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.
Sequestration of anti-platelet GPIIIa antibody in rheumatoid factor immune complexes of human immunodeficiency virus 1 thrombocytopenic patients.
Authors: Karpatkin S , Nardi M A , Hymes K B
Published: May 2006

Human immunodeficiency virus 1-related idiopathic thrombocytopenic purpura (HIV-1-ITP) patients have a 4-fold increased percentage of CD5+ B cells and a 4.8-fold increased percentage of serum immune complexes precipitated by polyethylene glycol (PEG-ICs) compared to control subjects, as reported previously. Since CD5+ B cells produce predominantly IgM rheumatoid factor (RF) vs. Fc of IgG and PEG-ICs contain high levels of IgM, we looked for the presence of RF in the immune complexes of HIV-1-ITP patients. PEG-ICs were adsorbed to protein A and dissociated with acid, and IgM and IgG were purified by gel filtration and affinity chromatography. Solid-phase ELISA was used to measure antibody specificity vs. platelets, Fc, and HIV-1 gp120, p24, and CD4. Dissociated IgG antibody reacted with platelets, HIV-1 gp120, p24, and CD4, but not with Fc. Serum IgG did not react with platelets or Fc but did react with HIV-1 gp120, p24, and CD4. Both PEG-IC IgM and serum IgM reacted with Fc as well as the other four antigens. Control IgM and IgG were unreactive. Isolated IgM from PEG-ICs relocated approximately 50% of the IgG preincubated with IgM to the Vo region of a G200 gel-filtration column. Anti-platelet IgG but not IgM could be affinity-purified from fixed platelets. Both F(ab')2 fragments of anti-platelet IgG and the total PEG-IC bound to platelets in a saturation-dependent manner. F(ab')2 of anti-platelet IgG inhibited 50% binding of PEG-IC to platelets at an F(ab')2/complex ratio of 3:1 (wt/wt). Scatchard analysis revealed two classes of binding sites: high-affinity Kd values of 0.8-1.8 nM and lower-affinity Kd values of 6.6-12.3 nM with respective numbers of binding sites of 44,000-57,000 and 122,000-256,000 (n = 4). Anti-platelet IgG of 6/6 patients precipitated GPIIIa from platelet lysates of surface 125I-labeled platelets. Platelet count correlated inversely with anti-platelet IgG (r = -0.73; P < 0.01; n = 27). Thus, PEG-ICs of HIV-1-ITP patients contain IgM RF, which sequesters serum anti-platelet IgG containing anti-GPIIIa. Anti-platelet IgG contributes to binding of immune complexes to platelets and correlates with thrombocytopenia.
Combination gene therapy for liver metastasis of colon carcinoma in vivo.
Authors: Chen S H , Chen X H , Wang Y , Kosai K , Finegold M J , Rich S S , Woo S L
Published: May 2006

The efficacy of combination therapy with a "suicide gene" and a cytokine gene to treat metastatic colon carcinoma in the liver was investigated. Tumor in the liver was generated by intrahepatic injection of a colon carcinoma cell line (MCA-26) in syngeneic BALB/c mice. Recombinant adenoviral vectors containing various control and therapeutic genes were injected directly into the solid tumors, followed by treatment with ganciclovir. While the tumors continued to grow in all animals treated with a control vector or a mouse interleukin 2 vector, those treated with a herpes simplex virus thymidine kinase vector, with or without the coadministration of the mouse interleukin 2 vector, exhibited dramatic necrosis and regression. However, only animals treated with both vectors developed an effective systemic antitumoral immunity against challenges of tumorigenic doses of parental tumor cells inoculated at distant sites. The antitumoral immunity was associated with the presence of MCA-26 tumor-specific cytolytic CD8+ T lymphocytes. The results suggest that combination suicide and cytokine gene therapy in vivo can be a powerful approach for treatment of metastatic colon carcinoma in the liver.
Negative-acting factor and superantigen are separable activities of the mouse mammary tumor virus long terminal repeat.
Authors: Wintersperger S , Salmons B , Miethke T , Erfle V , Wagner H , Günzburg W H
Published: May 2006

The open reading frame contained within the long terminal repeat (LTR) of mouse mammary tumor virus encodes Naf, a negative regulator of transcription, as well as a superantigen activity, Sag, which causes the deletion of specific classes of T cells. In the present study, the effect of Naf expression on different promoters and the coding requirements for Naf and Sag have been investigated. Sag activity was found to require only sequences in the LTR, whereas sequences located within the gag gene were additionally required for functional Naf activity. Surprisingly, both the classic promoter and a recently described promoter located in the LTR can give rise to both functional Naf and Sag. Further analysis of Naf revealed that the downregulatory effect was mediated by sequences located in the LTR and that heterologous promoters were also affected by Naf.
Interleukin 1 alpha and tumor necrosis factor alpha stimulate autocrine amphiregulin expression and proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells.
Authors: Woodworth C D , McMullin E , Iglesias M , Plowman G D
Published: May 2006

Infection with multiple sexually transmitted agents has been associated with inflammation of the cervix and an increased risk of cervical cancer in women infected with human papillomaviruses (HPVs). Two proinflammatory cytokines, interleukin 1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha), inhibited proliferation of normal epithelial cells cultured from human cervix. In contrast, both cytokines significantly stimulated proliferation of cervical cell lines (5 of 7) immortalized by transfection with HPV-16 or -18 DNAs or lines derived from cervical carcinomas (7 of 11). Stimulation was dose dependent from 0.01 to 1.0 nM and was blocked by specific inhibitors, such as the IL-1 receptor antagonist or the TNF type 1 or 2 soluble receptors. Growth stimulation by IL-1 alpha or TNF-alpha was accompanied by a 6- to 10-fold increase in RNA encoding amphiregulin, an epidermal growth factor (EGF) receptor ligand. Recombinant human amphiregulin (0.1 nM) was as effective as IL-1 alpha or TNF-alpha in promoting proliferation. Monoclonal antibodies that blocked signal transduction by the EGF receptor or that neutralized amphiregulin activity prevented mitogenic stimulation by IL-1 alpha or TNF-alpha. These studies indicate that IL-1 alpha and TNF-alpha stimulate proliferation of immortal and malignant cervical epithelial cells by an EGF receptor-dependent pathway requiring autocrine stimulation by amphiregulin. Furthermore, they suggest that chronic inflammation and release of proinflammatory cytokines might provide a selective growth advantage for abnormal cervical cells in vivo.
Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.
Authors: Wang S , Lee R J , Cauchon G , Gorenstein D G , Low P S
Published: May 2006

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.
Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.
Authors: Wetzler M , Talpaz M , Yee G , Stass S A , Van Etten R A , Andreeff M , Goodacre A M , Kleine H D , Mahadevia R K , Kurzrock R
Published: May 2006

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.
Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.
Authors: Takeuchi M , Baichwal V R
Published: May 2006

Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is involved in trafficking of lymphocytes to mucosal endothelium. Expression of MAdCAM-1 is induced in the murine endothelial cell line bEnd.3 by tumor necrosis factor alpha (TNF-alpha), interleukin 1, and bacterial lipopolysaccharide. Here we show that TNF-alpha enhances expression of a firefly luciferase reporter directed by the MAdCAM-1 promoter, confirming transcriptional regulation of MAdCAM-1. Mutational analysis of the promoter indicates that a DNA fragment extending from nt -132 to nt +6 of the gene is sufficient for TNF-alpha inducibility. Two regulatory sites critical for TNF-alpha induction were identified in this region. DNA-binding experiments demonstrate that NF-kappa B proteins from nuclear extracts of TNF-alpha-stimulated bEnd.3 cells bind to these sites, and transfection assays with promoter mutants of the MAdCAM-1 gene indicate that occupancy of both sites is essential for promoter function. The predominant NF-kappa B binding activity detected with these nuclear extracts is a p65 homodimer. These findings establish that, as with other endothelial cell adhesion molecules, transcriptional induction of MAdCAM-1 by TNF-alpha requires activated NF-kappa B proteins.
Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.
Authors: Kaipainen A , Korhonen J , Mustonen T , van Hinsbergh V W , Fang G H , Dumont D , Breitman M , Alitalo K
Published: May 2006

We have recently cloned the human fms-like tyrosine kinase 4 gene FLT4, whose protein product is related to two vascular endothelial growth factor receptors FLT1 and KDR/FLK1. Here the expression of FLT4 has been analyzed by in situ hybridization during mouse embryogenesis and in adult human tissues. The FLT4 mRNA signals first became detectable in the angioblasts of head mesenchyme, the cardinal vein, and extraembryonally in the allantois of 8.5-day postcoitus (p.c.) embryos. In 12.5-day p.c. embryos, the FLT4 signal decorated developing venous and presumptive lymphatic endothelia, but arterial endothelia were negative. During later stages of development, FLT4 mRNA became restricted to vascular plexuses devoid of red cells, representing developing lymphatic vessels. Only the lymphatic endothelia and some high endothelial venules expressed FLT4 mRNA in adult human tissues. Increased expression occurred in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. Our results suggest that FLT4 is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. They also support the theory on the venous origin of lymphatic vessels.
CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.
Authors: Boger D L , Johnson D S
Published: May 2006

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.
Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.
Authors: Harris R A , McQuilkin S J , Paylor R , Abeliovich A , Tonegawa S , Wehner J M
Published: May 2006

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.
Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases.
Authors: Egelman E H , Yu X , Wild R , Hingorani M M , Patel S S
Published: May 2006

Most helicases studied to date have been characterized as oligomeric, but the relation between their structure and function has not been understood. The bacteriophage T7 gene 4 helicase/primase proteins act in T7 DNA replication. We have used electron microscopy, three-dimensional reconstruction, and protein crosslinking to demonstrate that both proteins form hexameric rings around single-stranded DNA. Each subunit has two lobes, so the hexamer appears to be two-tiered, with a small ring stacked on a large ring. The single-stranded DNA passes through the central hole of the hexamer, and the data exclude substantial wrapping of the DNA about or within the protein ring. Further, the hexamer binds DNA with a defined polarity as the smaller ring of the hexamer points toward the 5' end of the DNA. The similarity in three-dimensional structure of the T7 gene 4 proteins to that of the Escherichia coli RuvB helicase suggests that polar rings assembled around DNA may be a general feature of numerous hexameric helicases involved in DNA replication, transcription, recombination, and repair.
Electrogenic Na/HCO3 Cotransporter (NBCe1) Variants Expressed in Xenopus Oocytes: Functional Comparison and Roles of the Amino and Carboxy Termini
Authors: McAlear Suzanne D. , Liu Xiaofen , Williams Jennifer B. , McNicholas-Bevensee Carmel M. , Bevensee Mark O.
Published: May 2006

Using pH- and voltage-sensitive microelectrodes, as well as the two-electrode voltage-clamp and macropatch techniques, we compared the functional properties of the three NBCe1 variants (NBCe1-A, -B, and -C) with different amino and/or carboxy termini expressed in Xenopus laevis oocytes. Oocytes expressing rat brain NBCe1-B and exposed to a CO2/HCO3− solution displayed all the hallmarks of an electrogenic Na+/HCO3− cotransporter: (a) a DIDS-sensitive pHi recovery following the initial CO2-induced acidification, (b) an instantaneous hyperpolarization, and (c) an instantaneous Na+-dependent outward current under voltage-clamp conditions (−60 mV). All three variants had similar external HCO3− dependencies (apparent KM of 4–6 mM) and external Na+ dependencies (apparent KM of 21–36 mM), as well as similar voltage dependencies. However, voltage-clamped oocytes (−60 mV) expressing NBCe1-A exhibited peak HCO3−-stimulated NBC currents that were 4.3-fold larger than the currents seen in oocytes expressing the most dissimilar C variant. Larger NBCe1-A currents were also observed in current–voltage relationships. Plasma membrane expression levels as assessed by single oocyte chemiluminescence with hemagglutinin-tagged NBCs were similar for the three variants. In whole-cell experiments (Vm = −60 mV), removing the unique amino terminus of NBCe1-A reduced the mean HCO3−-induced NBC current 55%, whereas removing the different amino terminus of NBCe1-C increased the mean NBC current 2.7-fold. A similar pattern was observed in macropatch experiments. Thus, the unique amino terminus of NBCe1-A stimulates transporter activity, whereas the different amino terminus of the B and C variants inhibits activity. One or more cytosolic factors may also contribute to NBCe1 activity based on discrepancies between macropatch and whole-cell currents. While the amino termini influence transporter function, the carboxy termini influence plasma membrane expression. Removing the entire cytosolic carboxy terminus of NBCe1-C, or the different carboxy terminus of the A/B variants, causes a loss of NBC activity due to low expression at the plasma membrane.
Cloacibacterium normanense gen. nov., sp. nov., a novel bacterium in the family Flavobacteriaceae isolated from municipal wastewater
Authors: Allen Toby D. , Lawson Paul A. , Collins Matthew D. , Falsen Enevold , Tanner Ralph S.
Published: May 2006

Phenotypic and phylogenetic studies were performed on three isolates of an unknown Gram-negative, facultatively anaerobic, non-motile, yellow-pigmented, rod-shaped organism isolated from raw sewage. 16S rRNA gene sequence analysis indicated that these strains were members of the Bergeyella–Chryseobacterium–Riemerella branch of the family Flavobacteriaceae. The unknown bacterium was readily distinguished from reference strains by 16S rRNA gene sequencing and biochemical tests. The organism contained menaquinone MK-6 as the predominant respiratory quinone and had a DNA G+C content of 31 mol%. A most probable number-PCR approach was developed to detect, and estimate the numbers of, this organism. Untreated wastewater from one plant yielded an estimated count of 1.4×105 cells ml−1, and untreated wastewater from a second plant yielded an estimated count of 1.4×104 cells ml−1. Signal was not detected from treated effluent or from human stool specimens. On the basis of the results of the study presented, it is proposed that the unknown bacterium be classified in a novel genus Cloacibacterium, as Cloacibacterium normanense gen. nov., sp. nov., which is also the type species. The type strain of Cloacibacterium normanense is strain NRS1T (=CCUG 46293T=CIP 108613T=ATCC BAA-825T=DSM 15886T).
MAGNIFYING COLONOSCOPY FOR THE DIAGNOSIS OF INFLAMMATORY CHANGES IN ULCERATIVE COLITIS
Authors: Sugano Satoshi , Fujinuma Sumio , Sakai Yoshihiro
Published: May 2006

Background:  Endoscopic observation is the most effective method for the evaluation of staging in ulcerative colitis (UC). However, in cases with very mild inflammatory activity, histopathological diagnosis may also be required. Unfortunately, biopsy‐related accidents are not uncommon. As an alternative, we have used a magnifying colonoscope commonly used for tumor diagnosis to examine in detail the colon mucosa of UC patients in clinical remission, and then compared these findings relative to conventional endoscopy using histopathological diagnosis.

Subjects and Methods:  Among UC cases examined by colonoscopy between April 2000 and April 2005, 27 cases without hematochezia for at least 1 month were enrolled in this study. Following observations of inflammatory changes using conventional colonoscopy, magnifying observation and biopsies at a total of 144 sites were evaluated. Using histopathological standards, acute‐phase inflammation was indicated by the presence of neutrophil infiltration, whereas chronic‐phase inflammation was indicated by infiltration of lymphocytes, plasma cells and eosinophils.

Results:  Indicators of significant inflammation by conventional observation was erosion. Under magnification, inflammation appears as superficial defects in mucosa and small whitish spots. When the presence of infiltrating neutrophils was used as a positive histological marker for inflammation, there was no difference in the accuracy of diagnosis by conventional observation (95.1%) versus magnifying observation (97.2%). In contrast, when lymphocyte infiltration was used as a marker, the accuracy of diagnosis increased significantly (88.2%) using magnifying observation relative to conventional observation (61.1%).

Conclusions:  Magnifying endoscopy can be used effectively in the evaluation of minute mucosal changes in cases of UC remission.
UNUSUAL FOREIGN BODY: ESOPHAGEAL IMPACTION AFTER CYANOACRYLATE INJECTION FOR GASTRIC VARICES
Authors: Cinar Kubilay , Onder Fatih Oguz , Nazligul Yasar , Dokmeci Abdulkadir , Ormeci Necati
Published: May 2006

Cyanoacrylate injection has been found in large series to be a remarkably safe and efficient treatment of gastric variceal bleeding. Dysphagia is a common local side‐effect of endoscopic intravariceal histoacryl injection. A 49‐year‐old woman with diagnosis of cirrhosis and portal hypertension related to hepatitis B virus infection was admitted to the Ibni Sina hospital due to dysphagia that started after injection of N‐butyl‐2‐cyanoacrylate (histoacryl) for gastric varices. She had undergone endoscopic gastric intravariceal histoacryl injection 2 years previously and again 1 month before the admission. There were no complications related to injection of the histoacryl or the endoscopy procedure itself. Dysphagia started immediately after the second endoscopy and was refractory to prokinetic and analgesic medication. She did not have any other complaint of medical significance. A diagnostic endoscopy revealed a heterogeneous foreign body stuck in the distal esophagus. Dysphagia resolved after endoscopic removal of the foreign body by endoscopic biopsy forceps. The heterogeneous material, which was mixed with food particles and tissue debris, was analyzed via infrared spectrophotometer and was found to be cyanoacrylate glue. In the present case, extrusion of the glue occurred earlier then expected and retching or vomiting might have dislodged the extruded glue from the stomach to the esophagus. If prolonged and intractable dysphagia starts after the endoscopy procedure, impaction of glue to the esophagus should be considered.
Determination of feeding mode in fishes: the importance of using structural and functional feeding studies in conjunction with gut analysis in a selective zooplanktivoreChirostoma estor estorJordan 1880
Authors: Ross L. G. , Martínez‐Palacios C. A. , Aguilar Valdez Ma. del C. , Beveridge M. C. M. , Chavez Sanchez Ma. C.
Published: May 2006

Anatomical and histological studies of the endangered atherinidChirostoma estor estorreveal that the species is ideally adapted to feeding on zooplankton. It has a superior protractile mouth with short unicuspid mandibular teeth. The buccal cavity is a highly adapted branchial sieve with branchial spines which develop in complexity with age to form a continuous flexible interdigitated mat. The filter bed has many of the characteristics of a cross‐flow filter, which is ideal for a continuously feeding and filtering animal as the filter bed will not readily become occluded. The aggregates from the cross‐flow filter pass to the rear of the buccal cavity where they are triturated by well‐developed pharyngeal teeth. The species has a short intestine (<0·3 × body length) with no histological evidence of stomach‐like structures, no pyloric caecae and with trypsin‐like enzymes operating at high pH. Feeding trials with natural plankton showed a sequence of particle size selection as the animals grow, with older animals taking cladocerans up to 700 μm in diameter. Although some adults occasionally take small fish prey, cumulatively, the present studies indicate that the fish is a zooplankton feeder throughout all its life stages.
Role of MAP Kinase‐Dependent Apoptotic Pathway in Innate Immune Responses and Viral Infection
Authors: Sumbayev V. V. , Yasinska I. M.
Published: May 2006

Mitogen‐activated protein (MAP) kinase cascades are multifunctional signalling networks that influence cell growth, differentiation, apoptosis and cellular responses to stress. Apoptosis signal‐regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c‐Jun N‐terminal kinases (JNK) and p38‐MAP kinase, which are responsible to induce apoptotic cell death. This pathway plays a pivotal role in the transduction of signals from different apoptotic stimuli. Recently, it has become evident that ASK1 and its downstream pathway are employed in the transduction of signals from Toll‐like receptors (TLR) – multistep processes that interfere with different intracellular signalling pathways. TLR are the key proteins that allow mammals to detect pathogens and mediate innate immune responses. In addition, ASK1 and its downstream pathway play a target role in the regulation of apoptosis in some cases of viral infection – AIDS, influenza, hepatitis C and others. In the present review, we summarize current knowledge about the role of ASK1 and its downstream pathway in innate immune responses and viral infection.
On the track of the Red Queen: bark beetles, their nematodes, local climate and geographic parthenogenesis
Authors: Meirmans S. , Skorping A. , Løyning M. K. , Kirkendall L. R.
Published: May 2006

Geographic parthenogenesis has been explained as resulting from parasite pressure (Red Queen hypothesis): several studies have found high degrees of sexuals where the prevalence of parasites is high. However, it is important to address whether prevalence of parasites mirrors risk of infection. We explored geographic parthenogenesis of Ips acuminatus bark beetles and their nematodes. Local climate is crucial for nematode stages outside the host, in spring and summer, and prevalence should thus be associated with those temperatures if prevalence reliably reflects exposure risk across populations. This was the case; however, high prevalence of a virulent nematode species was not associated with many sexuals, whereas highly sexual populations were characterized by high infection risk of benign nematodes. Low virulence of the latter makes Red Queen dynamics unlikely. Geographical patterns of parthenogenesis were instead associated with winter temperature and variance in temperature.