An anionic defensin from Plutella xylostella with potential activity against Bacillus thuringiensis.

Insect defensins, are cationic peptides that play an important role in immunity against microbial infection. In the present study, an anionic defensin from Plutella xylostella, (designated as PxDef) was first cloned and characterized. Amino acid sequence analysis showed that the mature peptide owned characteristic six-cysteine motifs with predicted isoelectric point of 5.57, indicating an anionic defensin. Quantitative real-time polymerase chain reaction analysis showed that PxDef was significantly induced in epidermis, fat body, midgut and hemocytes after injection of heat-inactivated Bacillus thuringiensis, while such an induction was delayed by the injection of live B. thuringiensis in the 4th instar larvae of P. xylostella. Knocking down the expression of nuclear transcription factor Dorsal in P. xylostella by RNA interference significantly decreased the mRNA level of PxDef, and increased the sensitivity of P. xylostella larvae to the infection by live B. thuringiensis. The purified recombinant mature peptide (PxDef) showed higher activity against Gram-positive bacteria, with the minimum inhibition concentrations of 1.6 and 2.6 µM against B. thuringiensis and Bacillus subtilis, respectively. To our knowledge, this is the first report about an anionic PxDef, which may play an important role in the immune system of P. xylostella against B. thuringiensis.

Apoptosis in neurodegenerative disorders: potential for therapy by modifying gene transcription.

Apoptotic, rather than necrotic, nerve cell death now appears as likely to underlie a number of common neurological conditions including stroke, Alzheimer's disease, Parkinson's disease, hereditary retinal dystrophies and Amyotrophic Lateral Sclerosis. Apoptotic neuronal death is a delayed, multistep process and therefore offers a therapeutic opportunity if one or more of these steps can be interrupted or reversed. Research is beginning to show how specific macromolecules play a role in determining the apoptotic death process. We are particularly interested in the critical nature of gradual mitochondrial failure in the apoptotic process and propose that a maintenance of mitochondrial function through the pharmacological modulation of gene expression offers an opportunity for the effective treatment of some types of neurological dysfunction. Our research into the development of small diffusible molecules that reduce apoptosis has grown from studies of the irreversible MAO-B inhibitor (-)-deprenyl. (-)-Deprenyl can reduce neuronal death independently of MAO-B inhibition even after neurons have sustained seemingly lethal damage. (-)-Deprenyl can also influence the process outgrowth of some glial and neuronal populations and can reduce the concentrations of oxidative radicals in damaged cells at concentrations too small to inhibit MAO. In accord with earlier work of others, we showed that (-)-deprenyl alters the expression of a number of mRNAs or of proteins in nerve and glial cells and that the alterations in gene expression/protein synthesis are the result of a selective action on transcription. The alterations in gene expression/protein synthesis are accompanied by a decrease in DNA fragmentation characteristic of apoptosis and the death of responsive cells. The onco-proteins Bcl-2 and Bax and the scavenger proteins Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD-2) are among the 40-50 proteins whose synthesis is altered by (-)-deprenyl. Since mitochondrial membrane potential correlates with mitochondrial ATP production, we have used confocal laser imaging techniques in living cells to show that the transcriptional changes induced by (-)-deprenyl result in a maintenance of mitochondrial membrane potential, a decrease in intramitochondrial calcium and a decrease in cytoplasmic oxidative radical levels. We therefore propose that (-)-deprenyl acts on gene expression to maintain mitochondrial function and decrease cytoplasmic oxidative radical levels and thereby reduces apoptosis. An understanding of the molecular steps by which (-)-deprenyl selectively alters transcription may lead to the development of new therapies for neurodegenerative diseases.

(-)-Deprenyl reduces neuronal apoptosis and facilitates neuronal outgrowth by altering protein synthesis without inhibiting monoamine oxidase.

(-)-Deprenyl stereospecifically reduces neuronal death even after neurons have sustained seemingly lethal damage at concentrations too small to cause monoamine oxidase-B (MAO-B) inhibition. (-)-Deprenyl can also influence the process growth of some glial and neuronal populations and can reduce the concentrations of oxidative radicals in damaged cells at concentrations too small to inhibit MAO. In accord with the earlier work of others, we showed that (-)-deprenyl alters the expression of a number mRNAs or proteins in nerve and glial cells and that the alterations in gene expression/protein synthesis are the result of a selective action on transcription. The alterations in gene expression/protein synthesis are accompanied by a decrease in DNA fragmentation characteristic of apoptosis and the death of responsive cells. The onco-proteins Bcl-2 and Bax and the scavenger proteins Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) are among the 40-50 proteins whose synthesis is altered by (-)-deprenyl. Since mitochondrial ATP production depends on mitochondrial membrane potential (MMP) and mitochondrial failure has been shown to be one of the earliest events in apoptosis, we used confocal laser imaging techniques in living cells to show that the transcriptional changes induced by (-)-deprenyl are accompanied by a maintenance of mitochondrial membrane potential, a decrease in intramitochondrial calcium and a decrease in cytoplasmic oxidative radical levels. We therefore propose that (-)-deprenyl acts on gene expression to maintain mitochondrial function and to decrease cytoplasmic oxidative radical levels and thereby to reduce apoptosis. An understanding of the molecular steps by which (-)-deprenyl selectively alters transcription may contribute to the development of new therapies for neurodegenerative diseases.

(-)-Deprenyl reduces PC12 cell apoptosis by inducing new protein synthesis.

(-)-Deprenyl, a monoamine oxidase (MAO)-B inhibitor, has been shown to increase neuronal survival and to alter protein synthesis and gene expression in astrocytic or PC12 cells independently of MAO-B inhibition. We used serum and nerve growth factor withdrawal to induce apoptotic death in PC12 cells to determine whether (-)-deprenyl increases neuronal survival by reducing apoptosis. (-)-Deprenyl reduced both cell death and internucleosomal DNA degradation in a concentration-dependent manner and was effective at concentrations too low to inhibit MAO (< 10(-9) M). (+)-Deprenyl did not increase PC12 cell survival, and, with the exception of pargyline, other MAO-A and MAO-B inhibitors did not alter apoptotic death. Transcriptional and translational inhibition showed that the reduction in apoptosis required the induction of new protein synthesis by (-)-deprenyl. Increased survival was induced if transcription was maintained for 4 h and translation for 6 h after (-)-deprenyl addition. The findings suggest that transcriptional induction may underlie the other MAO-independent actions of (-)-deprenyl.

(-)-Deprenyl alters the time course of death of axotomized facial motoneurons and the hypertrophy of neighboring astrocytes in immature rats.

(-)-Deprenyl previously was shown to increase the survival of rat facial motoneurons (FMns) after a loss of muscle-derived trophic support caused by axotomy at Postnatal Day 14 (P14) and to increase reactive astrogliosis after traumatic damage to the adult rat striatum. We estimated reactive astrogliosis in facial nuclei at 1, 3, 7, 14, and 21 days after transection of the facial nerve at P14 by two methods: first, by measuring the relative optical density (OD) of GFAP immunoreaction (GFAP-OD) in the facial nuclei and second by determining the relative area of GFAP immunoreactivity (GFAP-AREA) in the same nuclei. Both measures were taken for multiple immunoreacted sections through the length of each facial nuclei by using a control half section at the same brain stem level taken from an unlesioned, age-matched animal. The experimental and control facial nuclear half sections were coimmunoreacted using the "glued" half brain stem method. The facial nerve transections served to axotomize all of the FMns in the ipsilateral facial nuclei. The numbers of surviving FMns were examined at the same time points as above using counts of Nissl-stained somata from serial sections taken through each facial nucleus. We found that FMn loss occurred rapidly after axotomy in saline-treated animals and could be best fitted with a decaying exponential relationship (time constant 2.7 days). In the saline-treated animals, the FMn loss plateaued between 7 and 14 days at 74.8%, and 47% of the FMns were found to be lost within 3 days. Increases in the facial nuclear GFAP-OD values and GFAP-AREA values were evident as early as 1 day following axotomy (2.5 and 3.3 times normal, respectively) and reached maximal levels by 7 days (5.7 and 37.6 times normal, respectively). The administration of (-)-deprenyl slowed the loss of the FMns by 24-48 h (time constant 3.9 days) and increased the number of surviving FMns at 21 days by 2.1 times. Treatment with (-)-deprenyl was found to significantly increase GFAP-OD and GFAP-AREA at Day 1 by 71 and 32%, respectively, and at Day 3 by 22 and 27%, respectively. In contrast, it decreased GFAP-OD and GFAP-AREA by 42 and 19%, respectively, at Day 7, and by 20 and 12%, respectively at Day 21. Accordingly, as estimated by both measures, the drug increases reactive astrogliosis in the facial nucleus during the first 3 days after facial nerve transection and decreases the gliosis thereafter.(ABSTRACT TRUNCATED AT 400 WORDS)