Biochemical characterization, developmental expression, and induction of the immune protein scolexin from Manduca sexta.

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M(r) approximately 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M(r) approximately 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis.

In vivo distribution of immune protein scolexin in bacteria-injected Manduca sexta larvae.

The Manduca sexta larva-specific immune protein, scolexin, was isolated and (14)CH(3)-labelled by reductive alkylation. The influence of the bacterium Streptococcus faecalis on the hemocoelic distribution of the labelled scolexin was then analyzed. During bacterial challenge, most of the scolexin signal was detected in association with the hemocyte aggregations and nodules which formed; in this respect the protein sometimes appeared to be associated with hemocytes which had phagocytized bacteria, while at other times it was most concentrated in the nodule-associated, and free, coagulum. Areas of high scolexin activity were sometimes detected at various sites on the surface of the fat body. The scolexin did not appear to bind directly to bacterial cells. Up to 24 hr following the injection of S. faecalis, the larvae were still carrying out the formation of nodules; unlike the nodules of the 3 and 6 hr intervals, the nodules observed at 21-24 hr were covered with an apparently humorally derived, coagular capsule.

A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta.

An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.

Antibacterial hemolymph proteins of Manduca sexta.

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study.

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.

Surface morphology of peritrophic membrane formation in the cabbage looper, Trichoplusia ni.

The development of the peritrophic membrane in the cabbage looper, Trichoplusia ni, was investigated by scanning electron microscopy. The development of this membrane is characterized by a series of events suggested by the observations to be (1) secretion of material among and above the microvilli of the midgut epithelial cells, (2) maturation of this material into a randomly cross-linked fibrous matrix, and (3) aggregation of amorphous materials in and within the matrix. The membrane, possessing small discontinuities, remains intact in the midgut, but shows gross damage by the time it is passed from the insect, surrounding the feces.

Action of douglas fir tussock moth larvae and their microflora on dietary terpenes.

A single type of bacterium, tentatively identified as a member of the genus Bacillus, was isolated from 2 of 20 midguts of Douglas fir tussock moth larvae being fed a diet of fir needles. No bacteria could be isolated from most midguts. Although spherically shaped bodies were present in the food bolus, these bodies, if microorganisms, could not be distinguished from spherical bodies associated with the plant tissue. The Douglas fir tussock moth dietary terpenes were altered during their passage through the insects, with two new terpenes being detected in the feces. One of these was identified as isoborneol. The relative significance of the insect and gut microflora with respect to terpene modification is unresolved. The well-established toxicity of terpenes may account for the near absence of common gut microflora in the insects.

Some effects of douglas fir terpenes on certain microorganisms.

The Douglas fir terpene alpha-pinene was shown to inhibit the growth of a variety of bacteria and a yeast. Other terpenes of the Douglas fir, including limonene, camphene, and isobornyl acetate, were also inhibitory to Bacillus thuringiensis. All terpenes were inhibitory at concentrations normally present in the fir needle diet of Douglas fir tussock moth larvae. The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of B. thuringiensis spores for the Douglas fir tussock moth larvae. The terpene alpha-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.

Virulence of cloned variants of Autographa californica nuclear polyhedrosis virus.

The relative virulence of five different genotypic variants of Autographa californica nuclear polyhedrosis virus was tested by determining the 50% lethal dose of occluded virus for larvae of Trichoplusia ni. The 50% lethal dose values of uncloned virus and the five cloned genotypic variants ranged between 10 and 21 polyhedra per larva, and no statistically significant differences were observed. Cloning has therefore neither enhanced nor decreased the virulence of this potential microbial pesticide.

Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light.

The inactivation of Bacillus thuringiensis spores and spores treated with two protectants, one proteinaceous and the other a commercial product, Shade, at wavelengths of the near-ultraviolet and visible spectra and at 254 nm is described. Determination of the inactivating wavelengths may be used to establish an efficient sunlight protective system for B. thuringiensis when used as a microbial insecticide.

Specificity and genetics of S-adenosylmethionine transport in Saccharomyces cerevisiae.

The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine congruent to S-adenosyl-(5')-3-methyl-thiopropylamine greater than S-adenosylethionine greater than S-inosylmethionine greater than S-adenosylhomocysteins. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-insoyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as group important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.

Induction and repression in the S-adenosylmethionine and methionine biosynthetic systems of Saccharomyces cerevisiae.

Two methionine biosynthetic enzymes and the methionine adenosyltransferase are repressed in Saccharomyces cerevisiae when grown under conditions where the intracellular levels of S-adenosylmethionine are high. The nature of the co-repressor molecule of this repression was investigated by following the intracellular levels of methionine, S-adenosylmethionine, and S-adenosylhomocysteine, as well as enzyme activities, after growth under various conditions. Under all of the conditions found to repress these enzymes, there is an accompanying induction of the S-adenosylmethionine-homocysteine methyltransferase which suggests that this enzyme may play a key role in the regulation of S-adenosylmethionine and methionine balance and synthesis. S-methylmethionine also induces the methyltransferase, but unlike S-adenosylmethionine, it does not repress the methionine adenosyltransferase or other methionine biosynthetic enzymes tested.

Methionine adenosyltransferase and ethionine resistance in Saccharomyces cerevisiae.

The methionine adenosyltransferase is repressed in Saccharomyces cerevisiae during growth in the presence of excess methionine. The relationship of this repression to the level of intracellular S-adenosylmethionine is discussed. In conjunction with these studies, an ethionine-resistant mutant has been investigated which has a low level of methionine adenosyltransferase under all conditions tested. The mechanism of ethionine resistance in the latter strain apparently depends on its inability to form large quantities of intracellular S-adenosylethionine. With respect to the methionine adenosyltransferase, there is no apparent interaction between ethionine-resistant and ethionine-sensitive alleles when both are present in the heterozygous diploid.

Sai-1 mutation: saccharomyces cerevisiae: characteristics of inhibition by S-adenosylmethonine and S-adenosylhomocysteine and protection by methionine.

The relationship of methionine to the inhibition caused by S-adenosylmethionine and S-adenosylhomocysteine in strains containing the sai-1 mutation has been investigated and shown to affect indirectly the survival of the mutants. The ability of the mutants to take up both inhibitors is similar to that of the wild-type cells. The mutant also retains the ability to hydrolyze S-adenosylhomocysteine and incorporate the hydrolytic products into the various cellular fractions. Maximal inhibition of the sai-1 mutants occurs at an extracellular concentration of 0.005 mmS-adenosylmethionine and 0.025 to 0.05 mmS-adenosylhomocysteine when the cellular concentration is 0.05 mg (dry weight) per ml. The results suggest that the sai-1 mutation affects reaction(s) either not associated with methionine biosynthesis, or methionine synthesis and at least one other critical cellular function.

Transport of S-adenosylmethionine in Saccharomyces cerevisiae.

The properties of a specific system for the transport of S-adenosylmethionine in yeast are described. The process was pH-, temperature-, and energy-dependent, and showed saturation kinetics. The K(m) for the system was 3.3 x 10(-6)m. Of the S-adenosylmethionine moieties tested, only S-adenosylhomocysteine competitively inhibited the uptake of the adenosylsulfonium compound. Adenine, adenosine, methionine, homocysteine, and the sulfonium compound S-methylmethionine were without effect. The analogue S-adenosylethionine showed competitive inhibition. Under conditions of inhibition of protein synthesis by cycloheximide or methionine starvation, permease activity was stable. The mutant sam-p3 apparently was able to transport S-adenosylmethionine only by diffusion. Uptake by diploids containing this mutation was directly proportional to the gene dose.

Mutation of Saccharomyces cerevisiae preventing uptake of S-adenosylmethionine.

A mutant has been isolated whose aberration severely restricts the ability of cells of Saccharomyces cerevisiae to take up S-adenosylmethionine. The mutation apparently also affects adenosylhomocysteine uptake, but not that of the S-adenosylmethionine moieties adenine, homocysteine, homoserine, or methionine, nor the sulfonium compound, S-methylmethionine. It is a single, chromosomal mutation whose expression is not dependent on the presence of ammonium ions.

Dominant mutation for ethionine resistance in Saccharomyces cerevisae.

An ethionine-resistant mutant of Saccharomyces cerevisiae has been investigated whose mutation (et(r2)) confers resistance to the heterozygous diploid also containing the sensitive allele, et(s). The mutation is apparently specific for reversal of ethionine inhibition. The principal difference between the sensitive et(s) strain and the mutant was the latter's inability to concentrate large intracellular quantities of adenosylethionine. Reduced incorporation of ethyl groups or ethionine in other cellular fractions of the mutant was also detected. The data show that the mutant has not lost the ability to form adenosylethionine. It is suggested that the mutant has an increased ability to hydrolyze this sulfonium compound after it has been synthesized. It is possible that some of the ethionine is detoxified before it can participate in protein or adenosylethionine synthesis. No mutant alteration in accumulation of ethionine from the medium was detected. In the presence of ethionine, the parental strain accumulated 25 times more adenosylethionine than did the mutant. However, with methionine, only twice as much adenosylmethionine was accumulated by the parental strain as by the mutant.

Some mutants of Saccharomyces cerevisiae inhibited by adenoylmethionine and adenosylhomocysteine.

These investigations have established the existence of a novel type of non-nutritional mutant (ai) which is inhibited in the presence of two naturally occurring cellular compounds. The inhibition is complete at an extracellular concentration at least as low as 0.05 mumole/ml of either adenosylhomocysteine or adenosylmethionine. It is suggested that adenosylhomocysteine is the true inhibitor. The ai mutants are phenotypically indistinguishable from the wild type in the absence of inhibitors. The results have shown that, if any direct effect on the methionine biosynthetic pathway exists, it is a secondary rather than the primary effect of the inhibitors. The ai mutation does not involve the loss of the adenosylmethionine (or methylmethionine): homocysteine methyltransferase. In addition, the ai mutants accumulate, maintain, and utilize adenosylmethionine and methionine in a manner similar to the parental strain. No genetic relationship could be detected between the ai-1 mutation and several different markers affecting methionine biosynthesis. The ai-1 mutation was also shown to be genetically recessive. Methionine partially reverses the inhibition caused by adenosylmethionine or adenosylhomocysteine. Neither methylmethionine nor homocysteine reversed the inhibition, which showed that the homocysteine methyltransferase cannot catalyze the synthesis of sufficient methionine under these conditions to simulate the effects of extracellularly supplied methionine. If adenine is present, methionine does not cause reversal of inhibition due to adenosylmethionine or adenosylhomocysteine. From the data presented, it is clear that the ai mutation involves some metabolic control mechanism, though the alteration does not appear to be associated primarily with the biosynthesis of methionine.