The establishment of cell lines from imaginal wing discs of Spodoptera frugiperda and Plodia interpunctella

Continuous cell cultures were established from imaginal wing discs of 2 Lepidoptera, Spodoptera frugiperda and Plodia interpunctella. The S. frugiperda line (IAL-SFD1) grows as multicellular vesicles and responds morphologically and biochemically to the insect hormone, 20-hydroxyecdysone. In contrast, the P. interpunctella cells (IAL-PID2) grow as attached monolayers of small spindle-shaped cells and do not appear to have specific responses to 20-hydroxyecdysone, although growth rates are slowed in these cells upon exposure to the hormone.     

Effects of ingested phytoecdysteroids on the growth and development of two Lepidopterous larvae

The effects of ingested or injected 20-hydroxyecdysone on silkworm larvae (Bombyx mori) including death without moulting, death following completion of promoted moulting, death during promoted moulting (ecdysis inhibition) and inhibition in growth with and without effects on moulting, are dependent upon the concentration of exogenous hormone, the precise developmental stage of the treated larvae, and the duration of exposure to the exogenous ecdysteroid. Comparisons of 20-hydroxyecdysone with other phytoecdysteroids in the silkworm and pink bollworm, Pectinophora gossypiella, show a similar but more potent effect induced by ponasterone A, while cyasterone causes an ‘antiecdysone’ effect.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Glucuronidation and sulfation of p-nitrophenol in isolated rat hepatocyte subpopulations. Effects of phenobarbital and 3-methylcholanthrene pretreatment

Parenchymal cells, isolated from untreated (control), phenobarbital(PB)-or 3-methylcholanthrene(3-MC)-treated rats, were separated into four subpopulations according to cell density, and glucuronidation and sulfation of p-nitrophenol (PNP) in the hepatocyte subpopulations were investigated. PB enhanced the glucuronidation almost 2-fold but not the sulfation, while 3-MC enhanced both glucuronidation (3-fold) and sulfation (2-fold) in the original cell suspensions. Some gradation trends were found in the conjugation activities among the hepatocyte subpopulations: In the control experiment, the extent of glucuronidation in four subpopulations was virtually the same but sulfation in high-density hepatocytes was slightly higher than in low-density ones. Both glucuronidation and sulfation were higher in low-density hepatocytes from PB-treated rats, though the gradation was very modest. Glucuronidation and sulfation tended to be slightly higher in middle-density hepatocytes in the 3-MC experiment. However, no definite correlation in conjugation activities vs. cell density, like those seen in cytochrome P-450s vs. cell density in the hepatocytes isolated from PB-treated rats, were found in the subpopulations from control or inducer-treated rats. Simultaneous studies on acetylation of p-aminobenzoic acid (PABA) revealed that the activities in the subpopulations were virtually the same and the inducers had little influence on the activity.     

Status of reduced glutathione in primary cultures of rat hepatocytes and the effect on conjugation of benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide

The intracellular level of reduced glutathione (GSH) and GSH conjugation have been investigated in primary cell cultures of hepatocytes isolated from control rats, phenobarbitone (PB) and 3-methylcholanthrene (MC) treated rats. The data demonstrate that in all cell cultures the GSH concentrations show a triphasic pattern: (i) within 1 h of culture an initial marked decrease to 50% of the levels found in fresh hepatocytes; (ii) recovery of GSH concentrations to above the levels observed in fresh cells. This occurs after 6 h in culture with control cells and after 10-24 h with cells from either PB or MC treated rats and was most prominent in cells from PB-treated rats. (iii) A slow decline to between 30 and 40 nmol GSH/mg protein from 24 to 96 h in culture. Synthesis of GSH was slower in cultured cells from PB treated rats and this was confirmed by the resynthesis rates when diethylmaleate (DEM) was used to deplete GSH. The formation of GSH conjugates with racemic 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was measured in control cells in suspension and after 3 and 24 h in culture. Despite the decrease in GSH concentrations observed between 1 and 4 h after culture, the conjugation rates were not decreased.     

Differentiation of various types of biological oxidation of nitrogen in organic compounds

The various types of nitrogen which occur in organic compounds and which are susceptible to biological oxidation are clearly divided into groups depending upon the pK_a, of the constituent nitrogen. The enzymatic processes which give rise to the N-oxidation products are reviewed by a consideration of species differences, age of animal, pH optima, influence of inducing agents, inhibitors and microsomal pretreatments, as well as the stereochemistry of the nitrogen atom.From the data collected, a concept is developed which suggests that all basic amines (group I) are oxidised by a flavine adenine nucleotide (FAD)-dependent enzyme system, whereas non-basic nitrogen-containing compounds (group III) are oxidised by a cytochrome P450-dependent system.It is further suggested that compounds of intermediary pK_a,i.e. between 1 and 7 (group II), may be substrates for both enzyme systems, which would yield the same products, but by different processes. The extent to which N-oxidation occurs in a species would therefore depend on the pK_a of the substrate and the amounts and ratio of the two enzymes present, a lower pK_a favouring oxidation by the cytochrome P450 system and a higher pK_a favouring oxidation by the FAD system.In a similar manner, it is suggested that the oxidation of aromatic heterocyclic amines depends upon the pK_a of the nitrogen, compounds having a low pK_a being preferentially metabolised by nitrogen oxidation.     

Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin-2 (IL-2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.     

Fibre length and gibberellins A1 and A20 are decreased in Eucalyptus globules by acylcyclohexanedione injected into the stem

Trinexapacethyl (TriEt), an acylcyclohexanedionetype inhibitor of gibberellin (GA) biosynthesis, was applied to 3-year-old Eucalyptus globules saplings by localised injection near the base of each stem. The objective was to alter cambial region GA levels and to study the effects on secondary xylem fibre development. Seven weeks later wood samples, with bark and cambial region intact, were removed 10 and 30 cm above the point of injection. Fusiform cambial cell dimensions were compared with those of fibre-tracheids in the most recently formed 100 um of secondary xylem. Increasing TriEt applications from 5 to 5 000 mg active ingredient significantly reduced average fibre length, and to a lesser extent average fusiform cambial cell length. Also reduced was the number of cells in the cambial zone and the number of differentiating fibres with primary walls. However, no trends were evident for changes in fibre diameter, the proportion of vessel elements or the ratio of cambial ray cells to fusiform cambial cells. Two gibberellins (GA₁ and GA₂₀), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in cambial region tissues by gas chromatographymass spectrometry using stable isotope labelled internal standards. Increasing TriEt application reduced both GA₁ and GA₂₀ levels. Effects on IAA and ABA were not significant, although their levels tended to be lower at the highest TriEt application rate. The elongation of secondary xylem fibres was positively correlated with higher levels of endogenous GA₁ (r_s= 0.74, P < 0.01) and GA₂₀ (r_s= 0.72, P < 0.01). These results support a causal role for GA₁ in cambial cell division. They are also consistent with the hypothesis that the elongation of differentiating secondary xylem fibres in woody an–giosperms is dependent on GA₁ levels in the cambial region.     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase