Selection and characterization of two monoclonal antibodies specific for the Aspergillus flavus major antigenic cell wall protein Aflmp1

Aspergillus flavus is a major fungal pathogen of plants and an opportunistic pathogen of humans. In addition to the direct impact of infection, it produces immunosuppressive and carcinogenic aflatoxins. The early detection of A. flavus is therefore necessary to diagnose and monitor fungal infection, to prevent aflatoxin contamination of food and feed, and for effective antifungal therapy. Aspergillus-specific monoclonal antibodies (mAbs) are promising as diagnostic and therapeutic reagents for the tracking and treatment of Aspergillus infections, respectively. However, A. flavus has a complex cell wall composition and dynamic morphology, hindering the discovery of mAbs with well-characterized targets. Here we describe the generation and detailed characterization of mAb5.52 (IgG2aκ) and mAb17.15 (IgG1κ), which bind specifically to the highly immunogenic cell wall antigen A. flavus mannoprotein 1 (Aflmp1). Both mAbs were generated using hybridoma technology following the immunization of mice with a recombinant truncated version of Aflmp1 (ExD, including the homologous CR4 domain) produced in bacteria. We show that mAb5.52 and mAb17.15 bind specifically to A. flavus and A. parasiticus cell wall fragments (CWFs), with no cross-reaction to CWFs from other fungal pathogens. Immunofluorescence microscopy revealed that both mAbs bind to the surface of Aspergillus hyphae and that mAb17.15 also binds to spores. The epitope for both mAbs is localized within the CR4 region of the Aflmp1 protein. These Aspergillus-specific mAbs may be useful for the early detection of fungal infection in food/feed crops, for serodiagnosis in patients with invasive aspergillosis caused by A. flavus infection and for the development of antibody-expressing disease-resistant crops.     

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.     

POR C of Arabidopsis thaliana: a third light- and NADPH-dependent protochlorophyllide oxidoreductase that is differentially regulated by light

During the sequencing of the genome of Arabidopsis thaliana a gene has been identified that encodes a novel NADPH-protochlorophyllide oxidoreductase (POR)-like protein (accession number AC 002560). This protein has been named POR C. We have expressed the POR C protein in Escherichia coli and have determined its in vitro activity. POR C shows the characteristics of a light-dependent and NADPH-requiring POR similar to POR A and POR B. The expression of the POR C gene differs markedly from that of the POR A and POR B genes. In contrast to the POR A and POR B mRNAs, the POR C mRNA has been shown previously to accumulate only after the beginning of illumination. In light-adapted mature plants only POR B and POR C mRNAs were detectable. The amounts of both mRNAs show pronounced diurnal rhythmic fluctuations. While the oscillations of POR B mRNA are under the control of the circadian clock, those of POR C mRNA are not. Another difference between POR B and POR C was found in seedlings that were grown under continuous white light. The concentration of POR C mRNA rapidly declined and soon dropped beyond the limit of detection, after these seedlings were transferred to the dark. On the other hand, POR B mRNA was unaffected by this light/dark shift. When seedlings were exposed to different light intensities, the amounts of POR B mRNA remained the same, while POR A and POR C mRNAs were modulated in an inverse way by these light intensity changes. POR A mRNA was still detectable in seedlings grown under low light intensities but disappeared at higher light intensities, while the mRNA concentration of POR C rose with increasing light intensities. These different responses to light suggest that the functions of the three PORs of Arabidopsis are not completely redundant, but may allow the plant to adapt its needs for chlorophyll biosynthesis more selectively by using preferentially one of the three enzymes under a given light regime.     

Isolation and functional analysis of a strong specific promoter in photosynthetic tissues

Constitutive promoters such as CaMV (cauliflower mosaic virus) 35S and nos (nopaline syn-thase) have been used extremely as useful tools in many plant transgenic researches. Because of lacking temporal and spatial regulation, constitutive promoters have a number of potential drawbacks in genetically improved crops[1]. For example, constitutive expression of viral capsid proteins in plants may increase the risk of transvapsidation or viral recombination to generate new strains of phytopathogen…     

Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain.

A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.     

A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs

We report the isolation of a novel soybean gene, Msg, which is highly expressed in developing soybean pods. The gene shows significant homology to a family of fruit- and flower-specific genes, designated the major latex protein (MLP) homologues, so far reported in only a few species and whose functions are unknown. The MLPs are more distantly related to a group of pathogenesis-related proteins (IPR or PR-10) whose functions are likewise unknown. This is the first report of a MLP homologue in a plant for which there is already an IPR-protein reported. We performed an analysis of the Msg promoter with 14 different promoter fragments ranging from 0.65 kb to 2.26 kb, fused to the uidA (GUS) gene. High transient expression was obtained with all the constructs upon particle bombardment in soybean and green bean pods. Stable Arabidopsis transformants were obtained with the Agrobacterium vacuum infiltration method. The promoter is fully active in Arabidopsis only in plants transformed with the 2.26 kb fragment promoter, expressing GUS in nectaries, nodes, short style and in guard cells of the silique, pedicel and stem but not in mature leaves. Surprisingly, the proximal 650 bp TATA-containing region cannot function on its own in Arabidopsis and can be deleted without a change in expression pattern in both Arabidopsis and soybean. Thus, tissue-specific regions of the complex Msg promoter reside in the distal 5 regions upstream of a dispensable TATA box in contrast to many examples of tissue-specific elements that reside much closer to the TATA box.     

Isolation and characterization of a calmodulin-like protein from the cyanobacterium Nostoc sp. PCC 6720

A 21-kDa novel polypeptide which possesses characteristics normally considered to be diagnostic of the calmodulin present in eukaryotic cells was isolated from the cyanobacterium Nostoc sp. PCC 6720. The major technique employed in the isolation of the polypeptide was ion-exchange chromatography on a Mono Q column. The 21-kDa polypeptide was shown: to activate pea NAD kinase in vitro, in a Ca²⁺ requiring reaction; to react with polyclonal antibodies raised against spinach calmodulin, but not with those raised against bovine brain calmodulin; and to exhibit a Ca²⁺ dependent shift in migration during SDS-PAGE.Abbreviations ATCC American Type Culture Collection - DCPIP 2,6-dichlorophenylindophenol - PBS Phosphate buffered saline     

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m⁵C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m⁵C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA^Cys and tRNA^Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.     

A novel isoform of ATPase c subunit from pearl millet that is differentially regulated in response to salinity and calcium

Vacuolar ATPases help in maintaining the pH of the vacuoles and thereby play a crucial role in the functioning of vacuolar sodium-proton antiporter. Though the various subunits that make V₁ and V₀ sector have been reported in plants their regulation is not understood completely. We have cloned three different isoforms of vacuolar ATPase subunit c (VHA-c) from Pennisetum glaucum with homologies among themselves varying from 38% to ∼73% at the nucleic acid level. Using real-time PCR approach we have shown that the three isoforms are regulated in a tissue-specific manner under salinity stress. While isoform III is constitutively expressed in roots and shoots and does not respond to stress, isoform I is upregulated under stress. Isoform II is expressed mainly in roots; however, under salinity stress its expression is downregulated in roots and upregulated in shoots. Tissue specific expression under salinity stress of isoform II was also seen after exogenous application of calcium. This study for the first time shows the presence of three isoforms of PgVHA-c and their differential regulation during plant development, and also under abiotic stress.     

Isolation and characterization of a rough microsomal fraction from rat kidney that is enriched in lysosomal enzymes

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM(2)) from the bulk of rough microsomal material in rat kidney because of its greater density. 2. The specific activities of five acid hydrolases in the RM(2) fraction were approximately one-half those of a purified lysosomal (L) fraction and 10- to 30-fold greater than those of an ordinary rough microsomal (RM(1)) fraction. 3. These special rough microsomes have a distinctive ultrastructure and electron-cytochemical properties. Their cisternal content resembles the matrix of lysosomes in that it is electron-dense, osmiophilic and plumbophilic and gives a positive reaction for acid phosphatase activity. 4. Polyacrylamide-gel electrophoresis of soluble proteins from the L fraction resolved nine anionic glycoproteins, most of which exhibit acid hydrolase activities (Goldstone & Koenig, 1970, 1973; Goldstone et al., 1971a). The most anionic glycoprotein is the acidic lipoglycoprotein of the lysosomal matrix (Goldstone et al., 1970). 5. Polyacrylamide-gel electrophoresis of soluble proteins from the RM(2) fraction resolved two cationic glycoproteins with acid hydrolase activities (Goldstone & Koenig, 1973) and an anionic glycoprotein with the same electrophoretic mobility as the lysosomal lipoglycoprotein, but without its lipid constituents or capacity to bind the basic fluorochrome Acridine Orange. These constituents are considered to be the precursors of the lysosomal glycoproteins.     

AtPLC2, a gene encoding phosphoinositide-specific phospholipase C,is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized.The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian -type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.