The clearance of purines from the haemolymph of the cecropia silkmoth

The clearance of adenine, caffeine, guanine, theophylline, and xanthine from the haemolymph of diapausing pupae and pharate adults of Hyalophora cecropia was studied. In all animals caffeine and theophylline persisted, while the other purine bases were cleared within a few days.     

Adenylate and guanylate cyclases of cecropia silkmoth fat body.

Adenylate and guanylate cyclases were assayed in silkmoth fat body homogenates by measuring the conversion of [α-³²P]nucleoside triphosphates to cyclic [³²P]nucleotides. Adenylate cyclase was dependent on dithiothreitol, required either Mg²⁺ or Mn²⁺ for activity, was activated by NaF, and inhibited by triton X-100. Guanylate cyclase was not dependent on dithiothreitol, was strictly dependent upon Mn²⁺, unaffected by NaF, and activated by triton X-100. Both cyclases had pH optima near 8.0 and were located chiefly in the particulate fraction of homogenates. Activities of both cyclases were maintained or elevated during the larval-pupal transformation and, in contrast to cyclic nucleotide phosphodiesterases, showed little decline in the early diapausing pupa.     

Synthesis and intracellular transport of protein by the colleterial gland of the cecropia silkmoth

The tubular portion of the colleterial gland of the cecropia silkmoth secretes protein which labels extensively with glycine and appears as a single peak when separated by acrylamide gel electrophoresis. The radioactive peak does not stain with protein stains or absorb at 280 nm, but is dispersed by Pronase digestion. Synthesis of the peak is sensitive to inhibition by puromycin and cycloheximide but is not sensitive to actinomycin or chloramphenicol inhibition. Amino acid analysis revealed a composition of 30% glycine, 33% aspartic acid, and insignificant amounts of aromatic amino acids; the molecular weight was calculated to be 160,000. Autoradiographic analysis revealed that nearly all the glycine incorporated is transported from the mature secretory cells, and the half-time of secretion is calculated to be 3.4 hr. The possible use of this product as a marker for biochemical differentiation is discussed.     

Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia

Fat body cells of silkmoth pupae (Hyalophora cecropia ) contain granules, showing a less dense outer zone and a denser, often crystalline, inner portion appear after cocoon spinning and increase until the larval-pupal ecdysis; more granules are formed in females than in males. Urate granules, appearing fibrous in internal structure, first form about the same time, but their accumulation is more gradual, and continues in the pupa. Both types have been isolated by centrifugation. Protein granules dissolve in buffers to yield proteins 1 and 2, with distinct electrophoretic and antigenic properties. These proteins have been isolated individually from pupal fat body extracts by using their different thermal stabilities in phosphate buffer containing MgCl2 and (NH4)2SO4, respectively, and purification was completed by gel chromatography. Protein 1 has a molecular weight of 480,000 and a subunit of 85,000 daltons, while protein 2 gives values of 530,000 and 89,000, respectively. Their amino acid compositions are similar but distinct. Proteins 1 and 2 accumulate in the hemolymph, beginning 3 days before spinning, reach maximal levels at spinning, and then decline in the hemolymph while granules are formed in the fat body, although the total hemolymph protein concentration does not decline at this time. It is concluded that the fat body of the late, feeding larva synthesizes two related "storage proteins" and secretes them in partially crystalline granules as protein reserves for metamorphosis.     

Synthesis of blood and cuticular proteins in late pharate adults of the cecropia silkmoth

Incorporation of tritiated leucine into blood and cuticular proteins of male Hyalophora cecropia was monitored during the final third of pharate adult development. We found no changes in specific activity of the total blood proteins, but there were conspicuous alterations in the banding pattern obtained by acrylamide gel electrophoresis. The specific activity of the cuticular protein extract decreased with age, but the cause of this decline is not obvious. These final stages of exocuticle formation do not involve the appearance or synthesis of new blood or cuticular proteins.     

The cDNA-structure of the prothoracicotropic hormone (PTTH) of the silkmoth Hyalophora cecropia.

The structure of the prothoracicotropic neurohormone (PTTH) of the silkmoth Hyalophora cecropia was elucidated at the cDNA level. The identified cDNA of 803 nt, which is over 90% identical with the corresponding part of the Samia cynthia ricini PTTH gene, encodes a preprohormone of 240 amino acids. Presence of proteolytic cleavage sites indicates that the preprohormone is split into a signal peptide, an intercalated peptide (64 residues), and the PTTH monomer (125 residues). Preprohormones of H. cecropia, S. c. ricini, Antheraea pernyi, and Bombyx mori diversified considerably in all these parts, indicating that the evolution of PTTH is unusually fast. Since a similarly rapid, and concerted evolution of the corresponding receptor is unlikely, the PTTH activity probably depends on the conservation of relatively few amino acids allowing proper molecular folding.     

Investigation of an Ortho-quinone isomerase from larval cuticle of the American silkmoth,Hyalophora cecropia

Insect cuticles catalyze the formation of N-acetylnorepinephrine (NANE) and N-β-alanylnorepinephrine (NBANE) from N-acetyldopamine (NADA) and N-β-alanyldopamine (NBAD), respectively. An enzyme, involved in the reaction, has now been isolated from fifth stage larval cuticle of Hyalophora cecropia and partially characterized. The enzyme alone has hardly any activity towards NADA, but together with diphenoloxidases [catechol oxidases (EC 1.10.3.1) or laccases (EC 1.10.3.2)] it will produce NANE as the main product from NADA, indicating that NADA-quinone is the actual substrate for the enzyme. The enzyme is presumably an ortho-quinone para-quinone methide isomerase, and formation of NANE is due to non-enzymatic addition of water to the quinone methide. The enzyme combination mushroom tyrosinase-cuticular isomerase has pH optimum at 5.5, and the optimal substrate concentration is about 10 mM NADA.Together with the endogenous cuticular diphenoloxidases the isomerase can account for the formation of NANE observed when pieces of intact cuticle are incubated with NADA, and for the presence of NANE and NBANE in sclerotized cuticle.The possible roles of the enzyme in sclerotization and defense reactions in insects are briefly discussed.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. II. Scale-forming and socket-forming cells

In wings of the giant silkmoth, Hyalophora cecropia, scale-forming and socket-forming cells are first observed on day four of pharate adult development. Scale-forming cells appear synthetically active when they are first observed, for their basal region is filled with huge stacks of polyribosome-studed lamellate endoplasmic reticulum, numerous Golgi complexes containing secretory vesicles and many elongated mitochondria. During later development, some of these organelles diminish in number. Neck and scale regions are predominantly filled with longitudinally oriented microtubules and microfibril bundles, suggesting that their function is one of transport rather than synthesis. The scales originate as finger-like projections of the cell body. They subsequently elongate, flatten out and deposit a cuticle which has a surface differing somewhat from that in other lepidopterans. It consists of longitudinal ridges (1.8-2.4 μ apart), transverse ribs (0.6-1.0 μ apart) and microribs (0.10-0.13 μ apart). Socket-forming cells produce a socket around the neck region of each scale-forming cell. The socket differentiates into several morphologically distinct regions: an apical fibrillar region, a ribosomal region, a mitochondrial-microtubular region and a basal fibrillar region. The absence of polyribosome-studded lamellae of endoplasmic reticulum and Golgi complexes suggests that its primary function is not biosynthesis but support and protection of the scale.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. I. Generalized epidermal cells

The ultrastructure of wing epidermis of the giant silkmoth, Hyalophora cecropia, was studied during pupal diapause and the first half of development to the adult. In diapause, the generalized epidermal cells are characterized by many free ribosomes, some vesicles and small lamellae of rough endoplasmic reticulum, some scattered short mitochondria and a few small Golgi complexes. During the early states of post-diapause development, before and after the time of apolysis (separation of the epidermis from the overlying cuticle), there is a marked increase in structures often associated with synthetic functions, such as polyribosomes, lamellate rough endoplasmic reticulum and Golgi complexes. On day five of post-apolysis development, just after the appearance of scale-forming and socket-forming cells, the generalized epidermal cells lay down the cuticulin layer of the adult cuticle. At this stage and later, the polyribosomes and lamellate rough endoplasmic reticulum decrease in abundance. Cell nuclei show three phases of temporary transition from predominantly lobed to predominantly round profile, which correspond to periods of reported DNA synthesis. Throughout this developmental process, therefore, there is good correlation of fine structure with changes in macromolecular synthesis recorded elsewhere.     

Acute changes in the response to peripheral leptin with alteration in the diet composition

Dietary induced obesity in rodents is associated with a resistance to leptin. We have investigated the hypothesis that dietary fat per se alters the feeding response to peripheral leptin in rats that were fed either their habitual high- or low-fat diet or were naively exposed to the alternative diet. Osborne-Mendel rats were adapted to either high- or low-fat diet. Food-deprived rats were given either leptin (0.5 mg/kg body wt ip) or saline, after which they were provided with either their familiar diet or the alternative diet. Food intake of rats adapted and tested with the low-fat diet was reduced 4 h after leptin injection, whereas rats adapted and tested with a high-fat diet did not respond to leptin. Leptin was injected again 1 and 5 days after the high-fat diet-adapted rats were switched to the low-fat diet. Leptin reduced the food intake on both days. In contrast, when low-fat diet-adapted rats were switched to a high-fat diet, the leptin inhibitory response was present on day 1 but not observed on day 5. Peripheral injection of leptin increased serum corticosterone level and decreased hypothalamic neuropeptide Y mRNA expression in rats fed the low-fat but not the high-fat diet for 20 days. The data suggest that dietary fat itself, rather than obesity, may induce leptin resistance within a short time of exposure to a high-fat diet.     

Adiponectin changes in relation to the macronutrient composition of a weight-loss diet

Adiponectin is an adipose-derived protein with beneficial metabolic effects. Low adiponectin is associated with obesity and related diseases. Significant weight loss increases adiponectin, reducing disease risk. This study compared the effects of two weight-loss diets with different macronutrient compositions on adiponectin. Eighty-one obese women in two cohorts were randomized to a low-fat (LF) or a low-carbohydrate (LC) diet. All subjects underwent equivalent weight-loss intervention, with weight and other measures assessed at baseline and after 6 (cohort I) or 4 (cohort II) months. Body fat was measured by dual energy X-ray absorptiometry. Adiponectin was measured by radioimmunoassay. Diet intake was assessed using 24-h recalls and 3-day diet records. Data were analyzed via t-tests and repeated-measures factorial ANOVA using time, diet, and replicate (cohort I vs. cohort II) as factors. Age, weight, body fat, BMI, adiponectin, and diet were similar at baseline. Following intervention, macronutrient composition of the diet was vastly different between the groups, reflecting the assigned diet. Both groups lost weight and body fat (P < 0.001), with effect in LC dieters greater than LF dieters (-9.1 kg vs. -4.97 kg weight, P < 0.05 and -5.45 kg vs. -2.62 kg fat, P < 0.001). Adiponectin increased in the LC (+1.92 mcg/ml, P < 0.01), but not the LF (+0.86 mcg/ml, P = 0.81), group. There was no correlation between weight loss and increase in adiponectin. These results confirm that diet-induced loss of weight and body fat is associated with increased adiponectin concentrations. This effect is evident with weight loss of 10% or more, and may be greater with LC diets.