Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Different point mutations in the met oncogene elicit distinct biological properties.

The MET proto-oncogene, encoding the tyrosine kinase receptor for HGF, controls genetic programs leading to cell growth, invasiveness, and protection from apoptosis. Recently, MET mutations have been identified in hereditary and sporadic forms of papillary renal carcinoma (PRC). Introduction of different naturally occurring mutations into the MET cDNA results in the acquisition of distinct biochemical and biological properties of transfected cells. Some mutations result in a high increase in tyrosine kinase activity and confer transforming ability in focus forming assays. These mutants hyperactivate the Ras signaling pathway. Other mutations are devoid of transforming potential but are effective in inducing protection from apoptosis and sustaining anchorage-independent growth. These Met(PRC) receptors interact more efficiently with the intracellular transducer Pi3Kinase. The reported results show that MET(PRC) mutations can be responsible for malignant transformation through different mechanisms, either by increasing the growth ability of cells or by protecting cells from apoptosis and allowing accumulation of other genetic lesions.-Giordano, S., Maffe, A., Williams, T. A., Artigiani, S., Gual, P., Bardelli, A., Basilico, C., Michieli, P., Comoglio, P. M. Different point mutations in the met oncogene elicit distinct biological properties.     

Fluorine-substituted dihydrobicyclomycins: synthesis and biochemical and biological properties

Many studies show that selective introduction of fluorine within pharmacological agents leads to improved activities. In this study, we determine the effects of aryl fluorine substitution in 5a-(benzylsulfanyl)-dihydrobicyclomycin (3) on the in vitro inhibition of Escherichia coli rho-dependent ATPase activity. Compound 3 is an analog of bicyclomycin (1), which is the only known selective inhibitor of rho, and 1 and 3 have comparable in vitro inhibitory activities. We demonstrate that aryl fluorine substitution of 3 leads to increase in inhibitory activity but that the beneficial effects due to fluorine were dependent upon the site and number of fluorine substituents. The bioactivities are rationalized in terms of the bond moment created by the aryl fluoride bond within the 5a-aryl dihydrobicyclomycin-rho-binding pocket. Use of this hypothesis led to the design of dihydrobicyclomycin derivatives with superior in vitro rho inhibitory activities.     

Angiotensin-converting enzyme inhibitors: biochemical properties and biological actions

The review will cover the chemistry and biochemistry of angiotensin-converting enzyme inhibitors with emphasis on data published since the publication of previous reviews. The relative merits of each contribution will be evaluated, as well as their potential for leading to new discoveries. The biology of angiotensin-converting enzyme inhibitors will be brought up-to-date to give the reader an appreciation of the medical implications of this new type of antihypertensive agent.     

Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant

Summary It has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at theCh2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P 0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 P 0.1) tumor:liver ratios at 24, 72 and 168 h using¹¹¹In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.     

Synthesis and biological properties of chimeric interferon-alpha2b peptides

We have previously reported the antiproliferative activity of synthetic sequences 29-35 and 122-139 of the interferon-alpha2b (IFN-alpha2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-alpha2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on (125)I-IFN-alpha2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-alpha2b peptides behaved as partial agonists of the IFN-alpha2b molecule and may be of interest for drug design purposes.     

Structure and biological activity of human homologs of the raf/mil oncogene.

Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.     

Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow

Effective containment of gene flow in transgenic plants requires a promoter that is highly specific for male and female gametes or tissues. Here, we report the creation of a novel pollen-, stigma- and carpel-specific (PSC) promoter through the fusion of the pollen-specific LAT52 and carpel-specific AGL5 enhancers to a stigma-specific SLG promoter. Gene expression analysis showed that fusion of the LAT52 enhancer to the SLG promoter enables the latter to gain pollen-specific activity while the acquirement of carpel-specific activity requires the correct orientation of the inserted AGL5 enhancer in the PSC promoter, and only a forward- but not a reverse-oriented one is functional. The resulting fPSC promoter, when fused to DT-A, generated at least three aberrant gynoecium phenotypes. Type I plants exhibited shortened stigmatic tissues, resembling plants containing the DT-A gene controlled by the SLG promoter. However, type II and III plants displayed partial or complete ablation of gynoecia, and were unable to support the reproductive process. Type II and III plants also produced severely perturbed anthers and pollen in comparison to type I or SLG::DT-A plants, and transgenic pollen grains were unable, when out-crossed with control plants, to pass the transgene to the next generation in all plants examined, indicating that they are selectively eliminated. This tissue-specific ablation or perturbation is highly specific, and does not compromise vegetative growth. Evidently, the fPSC promoter faithfully acquires tissue specificity from the incorporated enhancers and promoter, and should have a practical application for transgene containment in non-fruit and -grain producing plant crops.     

Analysis of the structure-function relationship of tumour necrosis factor. Human/mouse chimeric TNF proteins: general properties and epitope analysis.

To analyse the structure-function relationship of tumour necrosis factor (TNF), a set of in-frame chimeric genes was constructed by coupling appropriate segments of the human and mouse TNF coding regions. Under control of the bacteriophage lambda inducible PL promoter high level expression of these chimeric genes was obtained in Escherichia coli. Although both human and mouse TNF were produced in E. coli as soluble proteins, a reduction of solubility was observed in some of the chimeric proteins. The specific activity was variable, but in some constructs comparable to human TNF, indicating that the structural conformation of these chimeric proteins resembled the human TNF structure. Neutralization analysis using two monoclonal antibodies directed against human TNF, indicated that the regions involved in the binding of these antibodies are distributed over multiple segments of the polypeptide. Further analysis by site-directed mutagenesis of one subregion allowed the identification of the Arg131 residue as involved in the binding of both neutralizing monoclonal antibodies; an Arg131----Gln replacement abolished antibody binding but did not affect the specific activity of TNF.     

The biological and biochemical properties of L-canaline, a naturally occurring structural analogue of L-ornithine.

Many leguminous plants synthesize L-canavanine and sequester this nitrogen-rich, non-protein amino acid in the seed (1,2). Arginase-mediated hydrolytic cleavage of L-canavanine, in a manner analogous to L-ornithine and urea formation from L-arginine, produces urea and L-canaline (3,4). The resulting canaline is distinctive in being the only naturally occurring amino acid which possesses the aminooxy group (Fig. 1). Canaline decomposes in several organic solvents employed for its analysis by partition and ion-exchange chromatography (5,6) but the important question of the overall stability of this substituted hydroxylamine has not been investigated.     

Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene.

Because functionally significant substrates for the tyrosyl protein kinase activity of pp60v-src are likely to include membrane-associated proteins involved in normal growth control, we have tested the hypothesis that pp60v-src could phosphorylate and alter the signaling activity of transmembrane growth factor receptors. We have found that the epidermal growth factor (EGF) receptor becomes constitutively phosphorylated on tyrosine in cells transformed by the src oncogene and in addition displays elevated levels of phosphoserine and phosphothreonine. High-performance liquid chromatography phosphopeptide mapping revealed two predominant sites of tyrosine phosphorylation, both of which differed from the major sites of receptor autophosphorylation; thus, the src-induced phosphorylation is unlikely to occur via an autocrine mechanism. To determine whether pp60v-src altered the signaling activity of the EGF receptor, we analyzed the tyrosine phosphorylation of phospholipase C-gamma, since phosphorylation of this enzyme occurs in response to activation of the EGF receptor but not in response to pp60v-src alone. We found that in cells coexpressing pp60v-src and the EGF receptor, phospholipase C-gamma was constitutively phosphorylated, a result we interpret as indicating that the signaling activity of the EGF receptor was altered in the src-transformed cells. These findings suggest that pp60v-src-induced alterations in phosphorylation and function of growth regulatory receptors could play an important role in generating the phenotypic changes associated with malignant transformation.     

Evaluation of meat products from cloned cattle: biological and biochemical properties

Agricultural utilization of cloned livestock produced by nuclear transfer and their products for food will require public and governmental acceptance. A series of studies of properties of meat derived from cloned cattle was carried out to collect data for the safety assessment of cloned cattle products. Meat samples obtained from embryonic cloned, somatic cloned and non-cloned cattle were analyzed for chemical composition, as well as amino acids and fatty acids. Digestibility, allergenicity, and mutagenicity of meat were also examined. There were no significant differences in these properties among embryonic cloned, somatic cloned and non-cloned cattle. The analyses and tests revealed that there were no significant biological differences in meat from a non-cloned, an embryonic cloned, or a somatic cloned animal. A 14-week feeding trial in rats showed there were no abnormalities in body growth, general condition, locomotor activity, reflexes, sexual cycle, urinalysis, hematology, blood biochemistry, and histology. This study showed for the first time that the biological/biochemical properties of meat of cloned cattle are similar to those of non-cloned cattle.     

The outer membrane of Treponema pallidum: biological significance and biochemical properties

Rabbits infected intravenously with Treponema pallidum were not markedly febrile, and the pyrogenicity of treponeme preparations administered intravenously to rabbits was negligible. The antibiotic polymyxin B did not induce any ultrastructural changes on the treponemal surface and was not lethal (immobilizing) for T. pallidum, which was, however, highly susceptible to detergents such as SDS. Extraction of treponemes with Triton X-100 removed the outer membrane (despite the presence of Mg2+) as shown by electron microscopy, and solubilized a limited number of proteins detectable by SDS-PAGE, including a dominant antigen (47 kDal) demonstrated by immunoblotting. None of the proteins were heat-modifiable. Periodic acid-silver staining of polyacrylamide gels for carbohydrate together with protease K digestion did not demonstrate major carbohydrate components in whole treponemes, or in the Triton-soluble fraction. Surface iodination of intact treponemes revealed very little surface exposure of treponemal proteins, although a protein which co-migrated with host albumin was labelled and appeared to be associated with the treponemal surface. Many treponemal proteins were, however, labelled when iodination was done in the presence of Triton. These observations, indicate that the outer membrane of T. pallidum differs significantly from those of many Gram-negative pathogens.     

Differential regulation of glucose transporter isoforms by the src oncogene in chicken embryo fibroblasts.

The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.     

Biological and biochemical activities of a chimeric epidermal growth factor-Elk receptor tyrosine kinase.

Eph, Elk, and Eck are prototypes of a large family of transmembrane protein-tyrosine kinases, which are characterized by a highly conserved cysteine-rich domain and two fibronectin type III repeats in their extracellular regions. Despite the extent of the Eph family, no extracellular ligands for any family member have been identified, and hence, little is known about the biological and biochemical properties of these receptor-like tyrosine kinases. In the absence of a physiological ligand for the Elk receptor, we constructed chimeric receptor molecules, in which the extracellular region of the Elk receptor is replaced by the extracellular, ligand-binding domain of the epidermal growth factor (EGF) receptor. These chimeric receptors were expressed in NIH 3T3 cells that lack endogenous EGF receptors to analyze their signaling properties. The chimeric EGF-Elk receptors became glycosylated, were correctly localized to the plasma membrane, and bound EGF with high affinity. The chimeric receptors underwent autophosphorylation and induced the tyrosine phosphorylation of a specific set of cellular proteins in response to EGF. EGF stimulation also induced DNA synthesis in fibroblasts stably expressing the EGF-Elk receptors. In contrast, EGF stimulation of these cells did not lead to visible changes in cellular morphology, nor did it induce loss of contact inhibition in confluent monolayers or growth in semisolid media. The Elk cytoplasmic domain is therefore able to induce tyrosine phosphorylation and DNA synthesis in response to an extracellular ligand, suggesting that Elk and related polypeptides function as ligand-dependent receptor tyrosine kinases.