Krystyna Grzelak

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10 pg/ml and 1 ng/ml with limit of detection 5 pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.

Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause

When one-day-old, last instar Galleria mellonella larvae are exposed to 18 degrees C they enter diapause and cease further development for several months. During diapause a group of proteins (72-84 kDa) synthesized in the fat body and secreted into the hemolymph is markedly elevated. Partial sequencing of the N-terminus of two proteins from this group confirmed their identity with larval hemolymph proteins (LHP) belonging to the family of hexameric storage proteins. The expression of two Lhp genes of known sequence (Lhp76 and Lhp82) were monitored in both diapausing and non-diapausing individuals. The expression of both genes and subsequent synthesis of the proteins (LHP76 and LHP82) is maintained until at least 90-100 days of diapause.

Biosynthesis and degradation of juvenile hormone in corpora allata and imaginal wing discs of Galleria mellonella (L.)

Abstract The rate of juvenile hormone (JH) biosynthesis by corpora allata-corpora cardiaca complex (CA/CC) during two last larval instars of Galleria mellonella was analysed. The rate of biosynthesis reaches maxima at the beginning of the VIth and VIIth instars. It is markedly reduced before the last larval ecdysis and after the first day of the last larval instar. After passing the second day of the last larval instar CA/CC exhibits again an increased ability for the biosynthesis of JH. The JH esterase activity in CA/CC is very low at the beginning of last larval instar and rapidly increases after the first day of this instar. Beginning on the second day of last larval instar the rate of JH hydrolysis is always higher than the rate of JH synthesis in CA/CC. It is concluded that the secretion of JH by CA/CC is possible until the second day of the last larval instar. After this, JH-acid can be supplied by CA/CC to peripheral tissues. The imaginal wing discs of mobile prepupa exhibit the ability to methylate JH-acid. It is concluded that some elevations of JH titre in G. mellonella haemolymph after the second day of VIIth instar are due in part to JH-acid methyltransferase activity in the imaginal discs.

Immunosensor Incorporating Anti-His (C-term) IgG F(ab’) Fragments Attached to Gold Nanorods for Detection of His-Tagged Proteins in Culture Medium

Immunosensors based on gold electrodes (electrochemical) or gold discs (optical) modified with 1,6-hexanedithiol, gold nanorods and Anti-His (C-term) monoclonal antibody F(ab’) fragment are described. The antigen detected by the sensing platform is a recombinant histidine-tagged silk proteinase inhibitor (rSPI2-His6). Electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) techniques were used as methods for detection of the antigen. This approach allows to detect the antigen protein in concentration of 10 pg per mL (0.13 pM) of culture medium. The immunosensor shows good reproducibility due to covalent immobilization of F(ab’) fragments to gold nanorods layer.

The activity of acetylcholinesterase during development of the moth, Celerio euphorbiae.

Abstract Acetylcholinesterase activity and acetylcholine content both increase during the growth of larvae and become inversely correlated in pharate pupae and emerged pupae. Dramatic changes of ACh and AChE occur in the abdomen during the first 8 days after the larval-pupal ecdysis and are sex-dependent and do not involve the central nervous system.

Low molecular weight silk proteins in Galleria mellonella

Abstract Galleria cocoon proteins have been extracted by different solubilizing agents. Nine protein bands were observed by gel electrophoresis, with molecular weights ranging from 18 to 420 kD. Three silk proteins of 24, 29 and 30 kD were extracted only in the presence of β-mercaptoethanol, suggesting that they are covalently linked by disulfide bonds to the large fibroin. They are likely to be the products of the highly abundant mRNA of the posterior silk gland cells. In vitro translation analysis of this mRNA yielded 24, 29 and 30 kD proteins. Thus, as in Bombyx , the Galleria silk is composed of several subunits, including fibroin and low molecular weight polypeptides. However, the genes coding for fibroin or low molecular weight silk proteins in Bombyx and Galleria do not show nucleotide base homology.

The effect of 20-hydroxyecdysone on expression of genes coding for low molecular weight silk proteins of Galleria mellonella

Abstract The low molecular weight Galleria silk proteins of 24 and 29–30 kDa are coded by the 1100 nucleotides long mRNAs of the posterior silk gland (PSG) cells. The synthesis of these proteins starts during the first 2 days of the last instar when the endogenous ecdysteroid titre is very low. The synthesis of 24 kDa protein precedes the synthesis of 30 kDa protein. PSGs from day 1 last instar larvae are sensitive to exogenous 20-hydroxyecdysone (20-HE) in vitro at 0.5 μg/ml concentration causing an increase of 1100 nucleotides long mRNAs. The electrophoretic analysis of the in vitro labelling proteins indicates that 20-HE may induced the synthesis of 30 kDa protein. Juvenile hormone II in vitro suppresses the stimulatory effect of 20-HE. PSGs from day 3 last instar larvae are insensitive to exogenous 20-HE in vitro .

Hormonal regulation of the expression of two storage proteins in the larval fat body of the greater wax moth (Galleria mellonella).

During larval development of the greater wax moth, Galleria mellonella, genes of storage proteins LHP76 and LHP82 are tissue- and stage-specifically expressed. In this study, hormonal regulation of this expression has been investigated in vivo. Messenger RNAs of the juvenile hormone (JH-suppressible) Lhp82 gene are present only during the feeding period of the final larval instar, suggesting that a high level of JH during earlier stages prevents its expression and that a small rise in JH titer observed on day 8 of the final larval instar is responsible for the rapid shut-off of its transcription. Application of 1micro g of JH analog (fenoxycarb) specifically inhibits expression of Lhp82, whereas Lhp76 mRNAs remain at the same level. 20-hydroxyecdysone (20HE) does not exert any inhibitory effects on transcription of Lhp genes when injected in a dose of 0.5 or 1.5 micro g per individual, regardless of larval age. However, the same dose of 20HE significantly lowers the rate of LHPs synthesis within the fat body and completely blocks secretion of LHPs into the hemolymph. Therefore, we propose that 20HE inhibits the synthesis of storage proteins and their secretion without altering the level of mRNAs.

Stimulation of rna transcription by juvenile hormone in degenerating silk glands

RNA transcription in the silk glands of the wax moth, Galleria mellonella, decreases during cocoon spinning and particularly later in pharate pupae, when the glands begin to degenerate. Application of juvenile hormone to insects at those developmental stages enhances synthesis of polyadenylated RNA 4-6 times and to lesser extent synthesis of total RNA. The newly formed poly(A)RNA appears identical with that from the normally fully functioning and silk-producing glands: it contains the same predominant poly(A)RNA class and is equally readily translated in the wheat-germ system. The results indicate that the hormone restores the function of silk glands at the transcriptional level.

Polyadenylated RNA in diapausing and developing embryos of Bombyx mori

Abstract The polysome fraction from diapausing embryos of Bombyx mori contains polyadenylated RNA which does not show messenger activity in vitro. The enhanced synthesis and polyadenylation of RNA during development of the embryo results in the appearance of active mRNA in polysomes.