Comparing the secretory pathway in honeybee venom and hypopharyngeal glands

We provide insights into the secretory pathway of arthropod gland systems by comparing the royal jelly-producing hypopharyngeal glands and the venom-producing glands of the honeybee, Apis mellifera. These glands have different functions and different product release characteristics, but both belong to the class 3 types of insect glands, each being composed of two cells, a secretory cell and a microduct-forming cell. The hypopharyngeal secretory cells possess an extremely elongate tubular invagination that is filled with a cuticular structure, the end-apparatus, anchored against the cell membrane by a conspicuous series of actin rings. In contrast, venom glands have no actin rings, but instead have an actin-rich brush border surrounding the comparatively short and narrow end-apparatus. We relate these cytoskeletal differences to the production system and utilisation of secretions; venom is stored in a reservoir whereas royal jelly and enzymes are produced on demand. Fluorescence-based characterisation of the actin cytoskeleton combined with scanning electron microscopy of the end-apparatus allows for detailed characterisation of the point of secretion release in insect class 3 glands.     

Antibacterial activity and mechanism of a scorpion venom peptide derivative in vitro and in vivo

BmKn2 is an antimicrobial peptide (AMP) characterized from the venom of scorpion Mesobuthus martensii Karsch by our group. In this study, Kn2-7 was derived from BmKn2 to improve the antibacterial activity and decrease hemolytic activity. Kn2-7 showed increased inhibitory activity against both gram-positive bacteria and gram-negative bacteria. Moreover, Kn2-7 exhibited higher antibacterial activity against clinical antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA). In addition, the topical use of Kn2-7 effectively protected the skin of mice from infection in an S. aureus mouse skin infection model. Kn2-7 exerted its antibacterial activity via a bactericidal mechanism. Kn2-7 killed S. aureus and E. coli rapidly by binding to the lipoteichoic acid (LTA) in the S. aureus cell wall and the lipopolysaccharides (LPS) in the E. coli cell wall, respectively. Finally, the hemolytic activity of Kn2-7 was significantly decreased, compared to the wild-type peptide BmKn2. Taken together, the Kn2-7 peptide can be developed as a topical therapeutic agent for treating bacterial infections.     

E. coli minichromosome replication in vitro and in vivo: comparative analyses of replication intermediates

The process of replication of Escherichia coli minichromosomes was examined by following the intermediates formed in vitro and in vivo. Replication initiated on a supercoiled closed circular (CC) monomer, proceeded rapidly to a late but incomplete stage in polymerization (the LC form) in both systems, passed more slowly through a series of open and closed circular catenated dimers with varying extents of intertwining between the monomer units, and then yielded, after decatenation, the supercoiled CC monomer. The replication patterns of two different minichromosomes were similar, although the LC form and the multiply intertwined dimers were much more evident in the smaller pAL4 than in pAL2. The same basic replication scheme was seen in vitro and in vivo but completion of polymerization and processing of the dimers were slower in vitro. Some radioactivity was detected in OC monomer early during replication, consistent with occasional decatenation of LC structures to produce OC molecules which then completed replication to form CC molecules. However, progression to CC catenated dimers prior to formation of CC monomers represented the major replication pathway.     

Biosynthesis of enterobacterial common antigen in Escherichia coli. In vitro synthesis of lipid-linked intermediates

An in vitro system was developed to study the biosynthesis of enterobacterial common antigen (ECA). Membranes of Escherichia coli were found to possess an enzyme activity that catalyzes the transfer of UDP-N-acetyl-acetylglucosamine-1-phosphate from UDP-N-acetyl-glucosamine (UDP-GlcNAc) to an endogenous lipid acceptor according to the reaction UDP-GlcNAc + P-lipid----GlcNAc-PP-lipid + UMP. The lipid-linked product was tentatively identified as GlcNAc-pyrophosphorylundecaprenol (lipid I) based on a comparison of its chemical and chromatographic properties with those of authentic GlcNAc-pyrophosphorylundecaprenol. The enzyme was dependent on the presence of Mg2+ for activity, and the reaction catalyzed by the enzyme was totally inhibited by the antibiotic tunicamycin in both the forward and reverse directions. Incubation of membranes with both UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) and UDP-GlcNAc resulted in the conversion of lipid I to a more polar compound, lipid II. The synthesis of lipid II was dependent on prior synthesis of lipid I. Characterization of the saccharide moiety of lipid II resulted in the identification of this compound as ManNAcA-GlcNAc-pyrophosphorylundecaprenol.     

Biosynthesis of rat liver transhydrogenase in vivo and in vitro

The biosynthesis of pyridine dinucleotide transhydrogenase, a homodimeric inner mitochondrial membrane redox-linked proton pump, has been studied in isolated rat hepatocytes. Newly synthesized transhydrogenase, having an apparent molecular weight identical to the enzyme of isolated liver mitochondria, was selectively immunoprecipitated from detergent extracts of isolated hepatocytes which were labeled with [35S]methionine. That the enzyme is a nuclear gene product is indicated since 1) synthesis was inhibited by cycloheximide, but not by chloramphenicol and 2) no synthesis could be demonstrated in hepatocyte ghosts which are competent only in mitochondrial translation. In addition to the mature form of the enzyme, a species about 2000 daltons larger was also immunoprecipitated from pulse-labeled cells. The half-life of the larger form during a subsequent chase at 37 degrees C was about 2 min, whereas the mature form was not degraded. The relationship between the two forms of the enzyme was established by in vitro studies. A protein approximately 2000 daltons larger than mature transhydrogenase was immunoisolated from a rabbit reticulocyte lysate system programmed with sucrose gradient fractionated rat liver mRNA. This protein was converted to a species having the same size as mature enzyme after incubation with either intact rat liver mitochondria or a soluble matrix fraction derived from mitoplasts. These studies indicate that transhydrogenase is synthesized in the cytoplasm as a higher molecular weight precursor which is post-translationally processed to the mature protein by a soluble matrix protease during or after membrane insertion.     

Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro

This protocol details the optimal conditions to establish a long-term primary culture of secretory cells from the venom gland of the Bothrops jararaca snake. Furthermore, these conditions allow the production and secretion of venom into the culture medium. Snake venom is a rich source of active molecules and has been used for bioprospection studies. However, obtaining enough venom from snakes is a major obstacle. Secretory cells of venom glands are capable of producing active toxins. Therefore, a culture of secretory cells is a good in vitro system to acquire the venom of snakes without capturing the animal from the wild. The protocol described here provides a rapid (approximately 4 h) and reproducible means of producing sufficient amounts of snake venom for biological investigations.     

Hyperalgesic and edematogenic effects of Secapin-2, a peptide isolated from Africanized honeybee (Apis mellifera) venom

Honeybee stings are a severe public health problem. Bee venom contains a series of active components, including enzymes, peptides, and biogenic amines. The local reactions observed after envenoming include a typical inflammatory response and pain. Honeybee venom contains some well-known polycationic peptides, such as Melittin, Apamin, MCD peptide, Cardiopep, and Tertiapin. Secapin in honeybee venom was described 38 years ago, yet almost nothing is known about its action. A novel, variant form of this peptide was isolated from the venom of Africanized honeybees (Apis mellifera). This novel peptide, named Secapin-2, is 25 amino acid residues long. Conformational analyses using circular dichroism and molecular dynamics simulations revealed a secondary structure rich in strands and turns, stabilized by an intramolecular disulfide bridge. Biological assays indicated that Secapin-2 did not induce hemolysis, mast cell degranulation or chemotactic activities. However, Secapin-2 caused potent dose-related hyperalgesic and edematogenic responses in experimental animals. To evaluate the roles of prostanoids and lipid mediators in the hyperalgesia and edema induced by this peptide, Indomethacin and Zileuton were used to inhibit the cyclooxygenase and lipoxygenase pathways, respectively. The results showed that Zileuton partially blocked the hyperalgesia induced by Secapin-2 and decreased the edematogenic response. In contrast, Indomethacin did not interfere with these phenomena. Zafirlukast, a leukotriene receptor antagonist, blocked the Secapin-2 induced hyperalgesia and edematogenic response. These results indicate that Secapin-2 induces inflammation and pain through the lipoxygenase pathway in both phenomena.     

Biosynthesis of ecdysones in isolated prothoracic glands and oenocytes of Tenebrio molitor in vitro

Isolated prothoracic glands from Tenebrio larvae synthesize in vitro α-ecdysone, but not β-ecdysone from 4-¹⁴C-cholesterol. Isolated abdominal oenocytes from the larvae synthesize mainly β-ecdysone, but only little α-ecdysone. When prothoracic glands and oenocytes are cultured together, the α-ecdysone derived from the prothoracic glands is oxidized by the oenocytes to β-ecdysone. The newly synthesized hormones are not stored in the cells, but are secreted into the medium if sufficient amounts of non-labelled hormones are present. If no unlabelled hormones are added to the culture medium, the newly formed hormones are converted to a large extent into polar conjugates.     

In vivo and in vitro interaction of DnaK and a chloroplast transit peptide

Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.     

Substrate specificity in vivo and in vitro in the formation of stilbenes. Biosynthesis of rhaponticin

The biosynthesis of the stilbene glucoside rhaponticin (3,5,3′-trihydroxy-4′-methoxystilbene 3-O-β-d-glucoside), a constituent of rhubarb (Rheum rhaponticum), was localized in the rhizome. Acetate and various phenylpropane derivatives were tested as precursors in feeding experiments. p-Coumaric acid was more efficiently incorporated than isoferulic acid, resveratrol (3,5,4′-trihydroxystilbene) was found to be the best precursor of rhaponticin. In vitro, for the stilbene-synthesizing system an even higher selectivity in favor of the p-hydroxy compound was observed. When various cinnamoyl-CoA derivatives were tested, membrane-bound enzyme preparations from rhizome converted at pH 7.5p-coumaroyl-CoA into resveratrol whereas rhapontigenin was not formed from isoferuloyl-CoA. Caffeoyl-CoA was converted to astringenin, but with lower rates and at a more acidic pH. The stilbene skeleton is, therefore, synthesized from a phenylpropane moiety carrying a 4′-hydroxysubstitution, while further derivatization to the 3′-hydroxy-4′-methoxy structure takes place at the level of stilbenes.