Accumulation of glycine in the fat body of the silkworm, bombyx mori L. [31]

INVESTIGATIONS of intestinal transport of amino-acids in the locust1,2 and silkworm3,4 have shown no evidence for active accumulation in a transport from the insect gut of amino-acids. When glycine-2-14C was administered in vivo to fifth instar larvae of the silkworm, 96 per cent of the radioactivity was incorporated into various tissues within 1 h whereas in vitro only 19 per cent of the activity was transported by the mid-gut of silkworm (unpublished work). These results suggested that continued absorption of glycine by the intestine could be aided by a facilitated diffusion mechanism in which amino-acids are rapidly removed from the site of absorption either by accumulation into other tissues or by degradation. Although the insect fat body has been assigned both accumulatory and dissimilatory roles5, the mechanism of accumulation of amino-acids has not been investigated. Our present experiments show that the silkworm fat body possesses an efficient mechanism for accumulating glycine and that both the accumulation and the release of glycine are metabolically controlled.

Intestinal transport in the silkworm, Bombyx mori L., of amino acids from mixtures

The rate of absorption of amino acids from mixtures has been studied in the silkworm midgut by using an in vitro perfusion technique. The rates differ for individual amino acids. A characteristic absorption pattern is observed which is independent of the amino acid composition of the mixture used. The metabolic inhibitors dinitrophenol and cyanide have no effect on the amino acid transport from mixtures. Based on these results an energy-independent, carrier-mediated transport is postulated.

Lipid composition of the silkworm Bombyx mori L

The constituents of silkworm fat were studied in detail. An unsaturated fat with a high concentration of phospholipid was generally observed. Its iodine value increased during metamorphosis. The free fatty acid concentration likewise increased from the spinning larvae to the moth stage. Analyses of silkworm organs revealed that the fat body had the most fat and the least free fatty acids, whereas haemolymph contained the least fat. Silk glands contained the maximum phospholipid percentage. Stearic acid predominated in those tissues that had a high percentage of phospholipid. Stearic acid was the predominant saturated fatty acid in both the phospholipids and lecithin, and it accounted for 35–50 per cent of the free fatty acids of all the tissues. Q10 was the ubiquinone present; also found were ubichromenol and tocopherol. Results show that silkworm sterol may be cholesterol. Intestines contained the maximum quantities of sterol, ubiquinone, ubichromenol, and tocopherol. The composition of silkworm phospholipids varies considerably from those of other insects, but lecithin is comparable in its composition with lecithins of other animals. The phospholipids had with them a highly complexed protein along with a polysaccharide. In experiments with snake venoms unsaturated fatty acids were found to be predominantly released from silkworm lecithin.

Evidence for the inability of the intestinal mitochondria of the silkworm, Bombyx mori L.to oxidize fatty acids

Conditions for the preparation of mitochondria from silkworm intestines have been standardized. The inability of mitochondria to oxidize fatty acids has been demonstrated. Evidence for the absence of an inhibitor in the mitochondria has been obtained.

Detoxication of benzoate by glycine conjugation in the silkworm, Bombyx mori L

The role of hippuric acid formation as a mechanism for detoxication of benzoic acid in the silkworm has been investigated. Benzoate is inhibitory to the growth of the silkworm and excreted as hippuric acid, which is not toxic. Hippuric acid is not a normal constituent of excreta. Synthesis of hippuric acid has been shown to occur in the intestines of the silkworm. Hippuricase activity is present in the fat body and silk-gland tissue.

Alkaline and acid phosphatases of the silkworm, Bombyx mori L.

The variations in the activities of the alkaline and acid phosphatases of the silkworm, Bombyx mori, were studied in all stages of the life cycle. From hatching until the spinning stage a steady increase was recorded in the activity of both the enzymes followed with a conspicuous decrease at each moult. During the pupal stage the alkaline phosphatase was almost absent, whereas the acid phosphatase maintained a high and constant value. Increase or decrease of the activity of the enzymes during larval development was reflected in a decrease or increase in the acid-soluble phosphorus content. Acid phosphatase activity slowly increased from laying of the eggs to hatching of the larvae with a concomitant decrease in the acid-soluble phosphorus. Tissue analysis showed a high concentration of the alkaline enzyme in the intestines, but the haemolymph was almost free of both enzymes. Feeding of inorganic phosphate increased the alkaline enzyme in the intestines, whereas glucose had no effect on either of the enzymes in the intestines.

Blood proteins of the silkworm Bombyx mori L

The qualitative and quantitative aspects of the proteins of the silkworm blood were studied by the technique of agarophoresis. The blood of larvae at the final stage revealed the presence of six different protein zones. Considerable differences in the patterns were observed at different stages of growth. There was an increase in the total nitrogen of the blood up to the 5th instar and then came a sudden decrease in the one-day old pupae. Nitrogen concentration was at its highest in egg 1 stage and the electrophoretic pattern closely corresponded to the final larval pattern. Results indicate to the involvement of silk glands in the synthesis and breakdown of a protein designated as protein 5.

Chloromycetin in the nutrition of the silkworm Bombyx mori L.-III. Influence on gut weight, oxygen uptake, and glucose absorption

Administration of chloromycetin has been found to enhance the oxygen uptake of the gut of the silkworm. The possibility that this increase might have been due to a thinning of the gut wall has been ruled out since the reduction in gut weight set in much later. Although glucose ultilization by the gut has been found to be increased in vitro, increase in oxygen uptake has not been affected in the presence of glucose. The possibility of a hormonal stimulation has been discussed.

Chloromycetin in the nutrition of the silkworm Bombyx mori L.-III. Influence on gut weight, oxygen uptake, and glucose absorption

Administration of chloromycetin has been found to enhance the oxygen uptake of the gut of the silkworm. The possibility that this increase might have been due to a thinning of the gut wall has been ruled out since the reduction in gut weight set in much later. Although glucose ultilization by the gut has been found to be increased in vitro, increase in oxygen uptake has not been affected in the presence of glucose. The possibility of a hormonal stimulation has been discussed.

Chloromycetin in the nutrition of the silkworm Bombyx mori L.-II. Influence on digestion and utilization of protein, fat, and minerals

The nature and extent of the influence of chloromycetin on larval digestion and utilization of the principal dietary constituents-the proteins, fats, and minerals-was studied. The antibiotic was shown to influence favourably the utilization of all the constituents studied. The results have been discussed in the light of these and other findings.

Mechanism of glycine uptake by the silk glands of the silkworm Bombyx mori L.

The transport of glycine in vitro into the silk glands of the silkworm has been studied. Glycine accumulates inside the tissue to a concentration higher than that present outside, indicating an active transport mechanism. The kinetics of uptake show a biphasic curve and two apparent Km values for accumulation, 0.33 mM and 5.00 mM. The effect of inhibitors on the energy metabolism of glycine transport is inconclusive. Exchange studies indicate the existence of two pools inside the gland, one that is easily removed by exchange and osmotic shock, and the other which is not. The results obtained conform with the carrier model of Britten and McClure concerning the amino-acid pool in E. coli.

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

DNA Polymerase4 from the Silk Glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.