Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

When plasma from third instar larvae of the fleshfly, Sarcophaga barbarta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 65% to ammonium sulphate, and it was found that the 50% fraction contained 95% of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration.The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7 day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.o.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme.The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 50% fraction, 62% now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in vivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.     

The in vitro biosynthesis and secretion of glycerol by larval fat bodies of chilled Ostrinia nubilalis

Fat bodies from diapausing fifth-instar larvae of Ostrinia nubilalis were incubated in vitro at 5 or 23°C in Grace's medium and the glycerol contents of the organ and incubation medium determined. Fat bodies from diapausing larvae chilled 3 weeks at 5°C secreted glycerol into the medium at 5°C at a net rate of approx. 0.75 nmol/mg fat body dry wt/h for at least 96 h while the tissue levels remained essentially constant. Depending upon the experiment, from 6 to 15 times more glycerol was produced in 24 h at 5°C by these fat bodies than by those taken from diapausing unchilled larvae and incubated at either 5 or 23°C. A minimal chilling period of 10–12 days was recognized as necessary for chilled larval fat bodies to demonstrate rates of glycerol synthesis greater than those of unchilled larvae and the lag showed a temporal correlation with changes in haemolymph glycerol concentrations. These results suggest that this response to chilling by O. nubilalis is relatively slow. While incubation, at 23°C, of fat bodies from previously chilled larvae did not result in cessation of glycerol secretion, the rate of its appearance in the culture medium decreased during the 24-h incubation period. Although the ability of chilled fifth-instar larvae to accumulate glycerol is not dependent upon the diapause state results show that clearance of glycerol from the haemolymph by rewarmed O. nubilalis is related to diapause intensity.     

Studies on Tyrosinase in the Housefly

A natural activator occurring in the aged pupae of the housefly, Musca domestica Maquart, was partially purified by means of fractionation with ammonium sulfate and of lyophilization. The preparation of the activator without accompanying tyrosinase activity made it possible to devise a method for measuring its activity, i.e., a partially purified protyrosinase which contained no activator was incubated together with natural activator, and the formation of tyrosinase activity was followed either manometrically or colorimetrically. Effects of concentration of natural activator, pH and temperature on the activation of protyrosinase were studied, resulting in an assumption that the activation occurs catalytically. Inhibition of the activation by various chemical reagents were studies and it was found that sodium picryl sulfate, N-bromosuccinimide or iodine inactivated natural activator irreversibly, and thioglycol or monoiodoacetate inhibited considerably. However, these reagents have almost no effect on the potential activity of protyrosinase. As the results, it was presumed that natural activator is an enzyme and protyrosinase is its substrate. The mode of activation of protyrosinase by the activator is, however, still obscure.     

Control mechanisms of tanning in Corcyra cephalonica

Localisation of a prophenoloxidase in the cytoplasm of plasmatocytes was histochemically demonstrable in Corcyra cephalonica, by incubating the blood with different phenolic substrates. The activation of this latent enzyme was found to be controlled by a protein factor present in the cuticle. The haemolymph prophenoloxidase could be activated in vitro by prior incubation of the blood sample at 0°C. The treatment with NaCl, EDTA, or detergents did not cause any activation. The exposure of blood samples to gamma-rays, repeated freezing and thawing, or addition of the cuticular activator, brought about rapid disruption of the haemocytes causing a release of phenoloxidase and acceleration of melanization of the blood. The administration of α-ecdysone to the last instar larvae induced premature pupation and accentuated the activation of phenoloxidase without altering the level of enzyme activity. The possible regulatory mechanisms of tanning during the development of Corcyra have been discussed.     

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Studies on Tyrosinase in Housefly

Occurrence of protyrosinase in the prepupae of housefly, Musca vicina Maquart, and its activation were described. The prepupae possessing no appreciable tyrosinase activity could be separated from the other aged pupae by putting them into water. Homogenate prepared from the prepupae contains protyrosinase and has no activation system for the proenzyme. As has been reported by Ohnishi²⁾ a certain activator occurred naturally in the aged pupae apparently activates the protyrosinase in vitro. However, contrary to Ohnishi’s results³⁾ it was found that this protyrosinase can be activated by the treatment with sodium dodecyl sulfate in vitro.     

Autocatalytic activation of the proenzyme form of the C1s subunit of the first component of complement.

The autocatalytic activation of the proenzyme form of the Cls subunit of the first component of complement is reported for the first time. Incubation of the purified proenzyme at 37° and pH 7.4 results in the evolution of esterolytic activity according to a second-order autocatalytic rate law. The lag phase portion of the sigmoidal activation curve can be shortened either by increasing the proenzyme concentration or by addition of the activated Cls subunit.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

Bactericidal substance induced in the haemolymph of Sarcophaga peregrina larvae

Bactericidal activity induced in the haemolymph of Sarcophaga peregrina larvae was investigated. It was found that this activity was due to a protein with a molecular weight of less than 10,000. The activity was lost on heating the haemolymph at 70°C for 5 min and it was dialyzable. This substance killed both E. coli and B. subtilis when incubated with them at 30°C, but not at 0°C. The possibility that this substance is lysozyme was excluded.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Change in overwintering mechanism of the Cucujid beetle, Cucujus clavipes

Overwintering larvae of the Cucujid beetle, Cucujus clavipes, were freeze tolerant, able to survive the freezing of their extracellular body fluids, during the winter of 1978–1979. These larvae had high levels of polyols (glycerol and sorbitol), thermal hysteresis proteins and haemolymph ice nucleators that prevented extensive supercooling (the supercooling points of the larvae were ? 10°C), thus preventing lethal intracellular ice formation. In contrast, C. clavipes larvae were freeze suspectible, died if frozen, during the winter of 1982–1983, but supercooled to ～ ? 30°C. The absence of the ice nucleators in the 1982–1983 larvae, obviously essential in the now freeze-susceptible insects, was the major detected difference in the larvae from the 2 years. However, experiments in which the larvae were artifically seeded at ? 10°C (the temperature at which the natural haemolymph ice nucleators produced spontaneous nucleation in the 1978–1979 freeze tolerant larvae) demonstrated that the absence of the ice nucleators was not the critical factor, or at least not the only critical factor, responsible for the loss of freeze tolerance in the 1982–1983 larvae. The lower lethal temperatures for the larvae were approximately the same during the 2 winters in spite of the change in overwintering strategy.     

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, from tropical and temperate regions

Seasonal adaptations of populations of the southwestern corn borer, Diatraea grandiosella, obtained from south-central Mexico (19°N latitude) and southeast Missouri (37°N latitude) were compared. Day length and temperature were found to serve as environmental cues to programme the larval diapause of both populations, but different critical values were observed. The critical day length for diapause induction was about 13 hr light/day for Mexican larvae and about 15 hr light/day for Missouri larvae, and was relatively stable at 20 to 30°C. Mexican larvae displayed a less-intense diapause than did Missouri larvae. Some diapausing Mexican larvae maintained at 25 or 30°C pupated in about 15 days, regardless of the day length to which they were exposed. The rate of diapause development of Mexican larvae was high at day lengths between 14 hr and 16 hr, whereas that of Missouri larvae was accelerated at day lengths of 16 hr at 25 and 30°C. Diapause development of Mexican larvae was virtually unaffected by chilling at 10°C, whereas that of Missouri larvae continued at a low rate at 10°C. Selection of Mexican larvae for diapause showed that only four generations were needed to significantly increase the incidence of diapause.     

Acid activation of protyrosinase from Aspergillus oryzae: homo-tetrameric protyrosinase is converted to active dimers with an essential intersubunit disulfide bond at acidic pH

Aspergillus oryzae protyrosinase (pro-TY) has a unique feature that the proenzyme is activated under conditions of acidic pH. The pro-TY was inactive at pH 7.0. The latent enzyme was activated at pH 3.0, and was slightly activated by sodium dodecyl sulfate (SDS). The molecular masses of the pro-TY and acid-activated tyrosinase (acid-TY) were 266 and 165 kDa, respectively, as estimated by gel-filtration chromatography. The CD spectra showed that the tertiary and/or quaternary structure was changed after the acid activation. On the basis of these results, we deduce that the intersubunit polar interaction is disrupted at pH 3.0, and that the tetrameric pro-TY dissociates to dimers. Tryptophan fluorescence spectra and binding assay of 8-anilino-1-naphthalene sulfonic acid (ANS) suggested that hydrophobic amino acid residues of the active site were exposed to solvent after acid treatment. It was likely that Cys108 formed an intermolecular disulfide bond between the subunits of dimeric acid-TY. The dimerization of acid-TY involving the intermolecular disulfide bond is essential for the activity.     

Freeze-tolerance adaptations,including haemolymph protein and lipoprotein nucleators,in the larvae of the cranefly Tipula trivittata

The terrestrial overwintering larvae of the cranefly Tipula trivittata were freeze tolerant (able to survive the freezing of their extracellular body fluids) throughout the winter and spring of 1982–1983 until they pupated in mid-May. The larvae were most cold tolerant (24 h lower lethal temperatures of ?25 to ?30°C) in late January and early February. Sorbitol, at a maximal concentration of ～0.4 M, was the only polyol determined to be present at high levels and sorbitol accounted for most of the seasonal fluctuation in osmotic concentration. Haemolymph inorganic ion (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^?) concentrations did not vary seasonally.The supercooling points of the larvae remained constant at ?6 to ?7°C over the study period because of the presence of haemolymph ice nucleating factors. These ice nucleating factors consist not only of haemolymph proteins, as had been demonstrated previously in other insect species, but also lipoproteins.     

Cold-induced glycerol accumulation by Ostrinia nubilalis larvae is developmentally regulated

Ostrinia nubilalis larvae reared under both nondiapause and diapause-inducing conditions were chilled at 5°C for various periods and their haemolymph glycerol concentrations were measured enzymatically. The ability of fifth (final) instars to accumulate glycerol was dependent upon cold stress but not the diapause state. Furthermore this response was independent of any cold-induced release of cephalic or thoracic hormones. The capacity of O. nubilalis larvae to express cold-induced glycerol accumulation was found to require ecdysis from the fourth to fifth instar. Eggs as well as second, third and fourth instars were completely incompetent. These results indicate that, at the biochemical level, a specific developmental programme or sequence is required for O. nubilalis to demonstrate this response to cold stress.     

Evaluation of the single radial diffusion technique for detection of nuclear polyhedrosis virus (NPV) infection in Heliothis zea

The single radial diffusion test is an effective method for detection of nuclear polyhedrosis virus infection in Heliothis zea larvae. Virus antigens were detected in some instances 48 hr after the larvae were exposed to virus. Most larvae tested positively for virus antigens 72 hr after exposure to the virus. The tests could be read within 4 hr if the incubation temperature was 35°C, and within 24 hr at 22°C.     

Changes in free amino acid composition in haemolymph of larvae of the wax moth,Galleria mellon ella L., during cold acclimation

• 1.1. Free amino acids were analysed in the haemolymph of Galleria mellonella larvae by HPLC chromatography with o-phthaldialdehyde (OPA)-l-thio-β-d-glucose as derivatization agent.
• 2.2. Fourteen primary amino acids were detected among which glutamine, alanine, γ-aminobutyric acid (GABA) and glycine predominated and constituted 67.7% of the amino acids found.
• 3.3. The concentration of GABA increased significantly with the age of larvae entering the wandering phase and reached a maximum during metamorphosis.
• 4.4. Analysis of cold-acclimated larvae revealed a net increase of free primary amino acids from 96 to 151.8 μmol/ml during consecutive acclimation to 0°C within 20 days and to 205.4μmol/ml during cold shock injury at 0°C (3 hr).
• 5.5. The bulk of this increase was accounted for by alanine, glycine, phenylalanine and lysine.

     

Protease-mediated prophenoloxidase activation in the haemolymph of American cockroach Periplaneta americana

Prophenoloxidase has been successfully obtained from the haemolymph of the cockroach Periplaneta americana using cane sugar saline solution. The proenzyme was activated by various exogenously added proteases such as chymotrypsin, trypsin, subtilisin and thermolysin. Thermolysin was found to be the greatest activator, followed by chymotrypsin and subtilisin. Chymotrypsin activation showed a lag period when compared with the other proteases tested, indicating that activation by chymotrypsin followed an indirect path, whereas, subtilisin and thermolysin activated the proenzyme directly.Exogenously added protease inhibitor showed inhibition towards protease-mediated prophenoloxidase activation. Benzamidine inhibited chymotrypsin and trypsin activation, whereas soybean trypsin inhibitor inhibited trypsin. In situ inhibitor isolated from the haemocytes of Periplaneta americana inhibited the prophenoloxidase activation and showed evidence for the presence of a built-in inhibition system for the release of the components of the prophenoloxidase activating system of P. americana. Electrophoretic localization of activated phenoloxidase showed two bands, suggesting the dimeric condition of high mol. wt prophenoloxidase.     

Studies on Tyrosinase in the Housefly

The mode of activation of protyrosinase prepared from prepupae of the housefly, Musca domestica vicina Maquart by sodium dodecyl sulfate (SDS), was studied by measuring the occurrence of tyrosinase activity over wide ranges of SDS concentrations, pH values and temperatures, either manometrically or colorimetrically. With respect to the effect of SDS concentration upon the activation of protyrosinase, it was found that there was a certain range of ratios between the concentration of SDS and that of protyrosinase which is effective for the activation. It was observed that a narrow pH range between 7 and 8 is effective for the activation. Studies of the effect of temperature on the activation indicate that the activation occurs preferentially over a limited range of temperatures. Thus, effective activation evidently occurs only with the specific experimental conditions mentioned above. These conditions suggest that a limited conformational change in the protyrosinase molecule results in the formation of tyrosinase. Precise observance of experimental conditions is required for complete activation of protyrosinase with SDS.     

Hormonal regulation of disaggregation of cellular fragments in the haemolymph of Lucilia cuprina

In the haemolymph of newly emerged adult blowfly (Lucilia cuprina) there is a transient change in the number of circulating, filamentous cellular fragments during the first hours after eclosion. Approximately 1 hr after the fly emerges from its puparium there is a rapid rise in the number of fragments from an undetectable quantity to a maximum of 1·3 × 10⁷ fragments per ml 75 min after eclosion. The number of fragments drops to about 5 × 10⁵ fragments per ml 1 hr later.Release of the fragments from their aggregation site can be inhibited by ligaturing flies between head and thorax as they emerge from their puparia. An injection of blood taken from older flies reverses this inhibition. Appreciable fragment disaggregating hormonal activity (FDH) can be detected in the haemolymph 5 min after eclosion.FDH and the tanning hormone, bursicon, could be the same chemical entity. Their release into the haemolymph is synchronous and both are proteins. The two hormonal activities cannot be separated from each other by ammonium sulphate fractionation or sieving through Sephadex G-50.