An invertebrate intestinal mucin (IIM) was identified from a lepidopterous insect, Trichoplusia ni. The IIM is a major protein constituent of the peritrophic membrane that facilitates the digestive process, as well as protecting invertebrate digestive tracts from microbial infections. The IIM demonstrated biochemical characteristics similar to vertebrate mucins, but exhibited strong association with the chitin-containing peritrophic membrane matrix. We have demonstrated that a baculovirus enhancin, which is encoded and carried by specific baculoviruses, has mucin-degrading activity both in vitro and in vivo. The in vivo degradation of IIM by enhancin was correlated with the enhancement of baculovirus infections in insects. These findings have shown that viruses have evolved a novel strategy to overcome intestinal mucinous barriers against microorganisms by utilizing a mucin-degrading enzyme.

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Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)

1 Administration of 3,4-methylenedioxymethamphetamine (MDMA or`ecstasy') to several species results in a long lasting neurotoxic degeneration of 5-hydroxytryptaminergic neurones in several regions of the brain. We have now investigated whether this degeneration is likely to be the result of free radicalinduced damage. 2 Free radical formation can be assessed by measuring the formation of 2,3-and 2,5-dihydroxybenzoic acid (2,3-DHBA and 2,5-DHBA) from salicylic acid. An existing method involving implantation of a probe into the hippocampus and in vivo microdialysis was modi®ed and validated. 3 Administration of MDMA (15 mg kg 71 , i.p.) to Dark Agouti (DA) rats increased the formation of 2,3-DHBA (but not 2,5-DHBA) for at least 6 h. Seven days after this dose of MDMA, the concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was reduced by over 50% in hippocampus, cortex and striatum, re¯ecting neurotoxic damage. There was no change in the concentration of dopamine or 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum. 4 p-Chloroamphetamine (PCA), another compound which produces a neurotoxic loss of cerebral 5-HT content, when given at a dose of 5 mg kg 71 also signi®cantly increased the formation of 2,3-DHBA (but not 2,5-DHBA) in the dialysate for over 4.5 h. post-injection starting 2 h after treatment. 5 In contrast, fen¯uramine administration (15 mg kg 71 , i.p.) failed to increase the 2,3-DHBA or 2,5-DHBA concentration in the dialysate. A single fen¯uramine injection nevertheless also markedly decreased the concentration of 5-HT and 5-HIAA in the hippocampus, cortex and striatum seven days later. 6 When rats pretreated with fen¯uramine (15 mg kg 71 , i.p.) seven days earlier were given MDMA (15 mg kg 71 , i.p.) no increase in 2,3-DHBA was seen in the dialysate from the hippocampal probe. This indicates that the increase in free radical formation following MDMA is occurring in 5-HT neurones which have been damaged by the prior fen¯uramine injection. 7 Administration of the free radical scavenging agent a-phenyl-N-tert-butyl nitrone (PBN; 120 mg kg 71 , i.p.) 10 min before and 120 min after an MDMA (15 mg kg 71 , i.p.) injection prevented the acute rise in the 2,3-DHBA concentration in the dialysate and attenuated by 30% the long term damage to hippocampal 5-HT neurones (as indicated by a smaller MDMA-induced decrease in both the concentration of 5-HT and 5-HIAA and also the binding of [ 3 H]-paroxetine). 8 These data indicate that a major mechanism by which MDMA and PCA induce damage to 5-hydroxytryptaminergic neurones in rat brain is by increasing the formation of free radicals. These probably result from the degradation of catechol and quinone metabolites of these substituted amphetamines. In contrast, fen¯uramine induces damage by another mechanism not involving free radicals; a proposal supported by some of our earlier indirect studies. 9 We suggest that these di erent modes of action render untenable the recent suggestion that MDMA will not be neurotoxic in humans because ...

Peritrophic membranes (PMs) are an invertebrate-unique structure that lines the digestive tract, playing important roles in facilitating food digestion and providing protection to the gut epithelium. The importance of PMs in insects has been recognized ever since its presence was identified 200 years ago. In the last 5 years, significant progress towards understanding the PM molecular structure and the mechanism for PM formation has been made. Recent studies on Type 1 PMs from lepidopteran larvae have suggested a model for the PM molecular structure and formation. The important physiological functions of the PM suggest that PMs can be a significant structural target for insect control and the current understanding of the structure of lepidopteran larval PMs has provided us with potential opportunities for targeting the PM by various mechanisms.

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