Expression and characterization of bispecific single-chain Fv fragments produced in transgenic plants.

We describe the expression of the bispecific antibody biscFv2429 in transgenic suspension culture cells and tobacco plants. biscFv2429 consists of two single-chain antibodies, scFv24 and scFv29, connected by the Trichoderma reesi cellobiohydrolase I linker. biscFv2429 binds two epitopes of tobacco mosaic virus (TMV): the scFv24 domain recognizes neotopes of intact virions, and the scFv29 domain recognizes a cryptotope of the TMV coat protein monomer. biscFv2429 was functionally expressed either in the cytosol (biscFv2429-cyt) or targeted to the apoplast using a murine leader peptide sequence (biscFv2429-apoplast). A third construct contained the C-terminal KDEL sequence for retention in the ER (biscFv2429-KDEL). Levels of cytoplasmic biscFv2429 expression levels were low. The highest levels of antibody expression were for apoplast-targeted biscFv2429-apoplast and ER-retained biscFv2429-KDEL that reached a maximum expression level of 1.65% total soluble protein in transgenic plants. Plant-expressed biscFv2429 retained both epitope specificities, and bispecificity and bivalency were confirmed by ELISA and surface plasmon resonance analysis. This study establishes plant cells as an expression system for bispecific single-chain antibodies for use in medical and biological applications.     

Production of singlet oxygen by eosinophils activated in vitro by C5a and leukotriene B4.

Using the trans-methoxyvinylpyrene analogues of benzo[a]pyrene-7,8-dihydrodiol (MVP) as a singlet oxygen ((1)O2) chemiluminescence probe, we have demonstrated that guinea pig eosinophils release (1)O2 when activated with the physiological agonists C5a and leukotriene B4. This release, which occurs at agonist concentrations as low as 10(-7) M, occurs more rapidly than activation with phorbol ester (10(-6) M), is similar in level, but is more transitory. In addition, the release of (1)O2 occurs in the absence of added bromide ions and represents, we propose, an important feature of eosinophil-mediated inflammatory damage.     

Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E.coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Study of Adenosine A2 Receptors in Membrane Preparations from Optic Tectum of Chicks

Binding properties of the subtypes of adenosine A2 receptors in membrane preparations and the effects of adenosine receptor ligands on cAMP accumulation in slices from the optic tectum of neonatal chicks have been investigated. [³H]2-[4-(2-p-carboxyethyl)phenylamino]-5'-N-ethylcarboxaminoadenosine (CGS 21680), a selective ligand for adenosine A2a receptors, did not bind to optic tectal membranes, as observed with rat striatal membranes. CGS 21680 also did not induce cyclic AMP accumulation in optic tectum slices. However, 5'-N-ethylcarboxamidoadenosine (NECA), 2-chloro-adenosine or adenosine induced a 2.5- to 3-fold increase on cyclic AMP accumulation in this preparation. [³H]NECA binds to fresh non-washed-membranes obtained from optic tectum of chicks, displaying one population of binding sites, which can be displaced by NECA, 8-phenyltheophylline, 2-chloro-adenosine, but is not affected by CGS 21680. The estimated K_D value was 400.90 ± 80.50 nM and the B_max was estimated to be 2.51 ± 0.54 pmol/mg protein. Guanine nucleotides, which modulate G-proteins activity intracellularly, are also involved in the inhibition of glutamate responses by acting extracellularly. Moreover, we have previously reported that guanine nucleotides potentiate, while glutamate inhibits, adenosine-induced cyclic AMP accumulation in slices from optic tectum of chicks. However, the guanine nucleotides, GMP or GppNHp and the metabotropic glutamate receptors agonist, 1S,3R-ACPD did not alter the [³H]NECA binding observed in fresh non-washed-membranes. Therefore, the adenosine A2 receptor found in the optic tectum must be the adenosine A2b receptor which is available only in fresh membrane preparations, and its not modulated by guanine nucleotides or glutamate analogs.     

PCR amplification of the spliced leader gene for the diagnosis of trypanosomatid parasites of plants and insects in methanol-fixed smears

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.     

Processing of extracellular lipase of Lactobacillus plantarum: involvement of a metalloprotease

The hypothesis of a proteolytic involvement in the extracellular lipase processing of a strain of Lactobacillus plantarum was considered and tested, in vitro assays with acid proteases, cathepsin D and renin revealed that both did affect lipolytic activity positively. In vivo assays with growth in the presence of the protease inhibitors pepstatin, phenylmethylsulfonyl fluoride, transepoxysuccinyl-L-leucylamido-(4-guanidino)butane and ethylenediaminetetraacetic acid showed that ethylenediaminetetraacetic acid did affect the pattern of the proteins that possess lipolytic activity. Therefore, it is suggested that a metalloprotease is involved in the processing of the extracellular lipases of L. plantarum, although other proteases can also be important.     

Expression of mRNA encoding insulin‐like growth factors I and II by uterine tissues and placenta during pregnancy in the rat

The uterus and the placenta synthesize insulin‐like growth factors (IGFs) and insulin‐like binding proteins (IGFBPs). These growth factors are implicated in processes of proliferation and differentiation that occur in the uterus. To determine the patterns of expression of IGFs during rat pregnancy we used in situ hybridization with digoxigenin labeled probes on uterus from day 7 to day 16 of pregnancy. In early gestation days (7–8) both IGF mRNAs showed similar tissue distribution with relative abundance in the stroma and circular muscle layer. On days 11 and 12 expression for IGF‐I mRNA was found in the mesometrial decidua and metrial gland and in the ectoplacental cone while clear expression of IGF‐II mRNA could only be found in the latter. On days 13 and 14, expression for IGF‐I mRNA could be detected in the mesometrial decidua and metrial gland but no expression was observed for IGF‐II mRNA. A gradient of IGF‐I mRNA expression could be observed in the placenta on day 16, with the trophoblastic cells of the basal zone expressing the signal with stronger intensity than in the labyrinthine zone. For IGF‐II mRNA the highest expression was associated with the labyrinthine zone. Endovascular trophoblast was positive for both mRNAs. The spatial and temporal patterns of expression suggests a role for IGFs in the process of decidualization as well as in the establishment, growth and differentiation of the various trophoblast cells of the placenta. Mol. Reprod. Dev. 53:294–305, 1999. © 1999 Wiley‐Liss, Inc.     

Distinct thyroid hormone‐dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH‐dependent period of the cochlea

Analyzing the thyroid hromone (TH)‐dependent period of the inner ear, we observed that the presence of triiodothyronine (T3) between postnatal day 3 (P3) and P12 is sufficient for functional maturation of the auditory system. Within this short time period, an unusual transient TH‐dependent expression of nonneuronal neurotrophin receptors (NT‐R) trkB and p75^NGFR was observed in correlation with neuronal and morphogenetic processes. The availability of thyroid hormone was revealed to be invariably correlated with (a) a transient expression of full‐length trkB in TRα1‐, TRα2‐ and TRβ1‐expressing hair cells concomitant to the segregation of afferent fibers and the synaptogenesis of efferent fibers; and (b) a transient expression of p75^NGFR in TRα1‐ and TRβ1‐expressing great epithelia ridge cells in direct spatiotemporal correlation with the appearance of apoptotic cells and morphogenetic maturation of the organ. For the first time, these data suggest a TH dependency of the expression of neurotrophin receptors in nonneuronal cells. A potential role of these peculiar neurotrophin receptor expression for the conversion of the biological function of TH on innervation patterning and morphogenesis during the critical TH‐dependent period of the inner ear may be considered. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 338–356, 1999     

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.