Hydrolysis of juvenile hormone in diapausing and non-diapausing larvae of the southwestern corn borer,Diatraea grandiosella

Summary The role of juvenile hormone (JH) esterases in relation to the diapause state of the southwestern corn borer,Diatraea grandiosella, was examined. The facultative larval diapause of this insect is dependent upon the presence of JH. Plasma, fat body, midgut, and body wall extracts metabolized [³H]JH I and [³H]JH III to JH-acid in vitro. JH-diol, JH-acid-diol, or conjugated polar metabolites were not detected. A longer half life of [³H]JH I was found in vitro in the plasma of diapausing larvae than in that of non-diapausing larvae. Although JH hydrolytic activity was relatively low in the plasma of pre-diapausing and diapausing larvae, systematic changes were observed suggesting that JH esterases may be involved in regulating the JH titer during this period. The JH hydrolytic activity found in the plasma of diapausing larvae was 3 to 5 times lower than that found in the plasma of mid-last instar non-diapausing larvae. Gel filtration profiles obtained from the plasma of diapausing and non-diapausing larvae suggested that JH esterases and -naphthyl-acetate esterases are different enzymes. Multiple overlapping peaks of JH hydrolytic activity with an apparent molecular weight range of 43,000 to 75,000 were detected, whereas 2 separate peaks of -naphthyl-acetate hydrolytic activity (apparent mol. wt. ca. 54,000, and 120,000) were detected. Gel filtration of supernatants of fat body indicated that JH was hydrolyzed at a lower rate by the fat body of pre-diapausing larvae than by that of non-diapausing larvae.     

Ecdysone-stimulated secretion from the salivary glands of Manduca sexta

Metamorphosing larvae of Manduca sexta (Lepidoptera) secrete, during a brief interval, 1–2 ml of fluid which contains up to 2.5 mg of protein, including a 5000-dalton protein which appears at metamorphosis. The protein is secreted in the copious alkaline fluid (pH 9–10), which is released into the burrow. When salivary glands are isolated in vitro in Grace's medium, 20-hydroxyecdysone at first stimulates and then suppresses the synthesis and secretion of the protein.     

Brain neurosecretory cell and ecdysiotropin activity of the non-diapausing,pre-diapausing and diapausing southwestern corn borer, Diatraea grandiosella Dyar

The location and number of brain neurosecretory cells were studied in the larval southwestern corn borer. One posterior, two median and two lateral groups of paraldehyde-fuchsin positive cells were found in each cerebral hemisphere.Implantation of brain parts containing different groups of neurosecretory cells revealed that the median neurosecretory cells contained higher ecdysiotropic activity than the other cell groups. In vitro culture of ecdysial gland with brain or brain-parts extract showed also that the median neurosecretory cells contained much higher ecdysiotropic activity than other neurosecretory cells. To estimate the ecdysiotropic activity of pre-diapausing 6th instar larvae, their brain or brain extract was incubated in culture medium containing an ecdysial gland from a day-4 last-instar non-diapausing larva. Data showed that the ecdysiotropic activity in the pre-diapausing larvae was far lower than in non-diapausing and diapausing larvae.     

Kinetics of the co-operative and non-co-operative reaction of Helix pomatia haemocyanin with oxygen

The kinetics of the reaction of Helix pomatia haemocyanin with oxygen have been studied under conditions where ligand binding is co-operative (n = 4.5). The dissociation of oxygen from oxyhaemocyanin in the presence of sodium dithionite and the combination of deoxyhaemocyanin with oxygen were studied by the stopped-flow technique. The combination with oxygen, as well as the dissociation of oxyhaemocyanin, are clearly autocatalytic. The initial rate constant for oxygen combination to the fully deoxygenated state is 0.2 to 0.3 × 10⁶m^?1 s^?1; during the course of the reaction the rate constant increases to a value higher than 10⁶m^?1s^?1.The initial rate of oxygen dissociation from fully saturated haemocyanin is 10 s^?1, increasing to about 30 s^?1 as the reaction proceeds. Thus, both the combination and the dissociation rate constants contribute to the co-operativity of oxygen binding.Temperature-jump relaxation experiments were carried out at fractional oxygen saturations larger than 0.7. The dependence of the relaxation rate upon the concentration of the reactants indicates the presence of one principal bimolecular process. The calculated combination and dissociation rate constants for this process are: 3.8 × 10⁶m^?1 s^?1 and 10 s^?1, respectively. Evidence is presented which shows that the transition from the T-state to the R-state of the protein is relatively slow. Both the T and R-state seem to be largely stabilized at the expense of intermediate states.Under other conditions, where oxygen binding is non-co-operative, temperature-jump and stopped-flow experiments reveal considerable kinetic heterogeneity.     

Diapause of the southwestern corn borer, Diatraea grandiosella: Further evidence showing juvenile hormone to be the regulator

Further evidence is presented to demonstrate the involvement of juvenile hormone (JH) in regulating diapause in the final larval stage of the southwestern corn borer. Diatraea grandiosella. JH titres in the haemolymph were measured throughout the entire diapause period. Additional results showed that actively secreting corpora allata are necessary to maintain diapause because allatectomized larvae terminated diapause prematurely. A topical application of JH mimic 2 days after the allatectomy prevented this premature termination of diapause. Intact nervous connections between the brain and the corpora allata were necessary for the maintenance of JH secretion. Other surgical work showed that the brains of nondiapausing larvae exhibited a higher ecdysiotropic activity than those of pre-, early-or mid-diapausing larvae.A single application of a JH mimic was more effective in maintaining a diapause-like state in nondiapausing larvae than were repeated topical applications of C₁₈-JH or an implantation of active corpora allata, suggesting that JH was more rapidly metabolized than was the JH mimic. The oxygen consumption of diapausing larvae which had received repeated topical applications of JH mimic was not significantly elevated over that of the controls indicating that treated larvae maintained a low metabolic rate even though they reverted to the spotted morph. A single application of 0.03 μg JH mimic/larva was sufficient to prolong diapause, thereby confirming that JH is necessary for diapause maintenance.     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Limited proteolysis of liver and muscle aldolases: Effects of subtilisin,cathepsin B,and Staphylococcus aureus protease

Limited proteolysis of rabbit liver and muscle aldolases by subtilisin and cathepsin B results in decreased catalytic activity, associated with the release of acid-soluble peptides from the COOH terminus. Analysis of the sequence of these peptides confirms the COOH-terminal sequence of the muscle enzyme and provides new information on the COOH-terminal sequence of the liver enzyme. As previously reported for muscle aldolase, cathepsin B releases mainly dipeptides from the COOH terminus of liver aldolase. The COOH-terminal sequence of rabbit liver aldolase is SerThrGlnSerLeuPheThrAla SerTyrThrTyr. The Gln-Ser bond is resistant to Staphylococcus aureus protease, which hydrolyzes a GluSer bond at the corresponding positions in the muscle enzyme.     

Factors influencing the developmental capacity of Galleria larval epidermis

The nature of the cuticle secreted by integument from a day-1 penultimate instar larval Galleria when cultured in vivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses.     

Subunit structure of anthranilate synthase from Neurospora crassa: Preparation and characterization of a protease-free form

The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of M_r 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

Evolutionary and neurobiological implications of selective phonotaxis in the spring peeper (Hyla crucifer)

Males of the spring peeper (Hyla crucifer) in central Missouri produce frequency-modulated, sinusoidal advertisement calls with a duration of 90–250 ms, and a mid-point frequency of 2800–3360 Hz. The frequency of the call is inversely correlated with body size. In playback experiments with synthetic stimuli, females did not prefer a frequency-modulated call to a call of constant frequency. Females preferred a call with a duration of 150 ms to sounds with durations of 40, 75 and 400 ms; a call of 300 ms was just as attractive as the 150-ms call. Females preferred a call of 2875 Hz to alternatives of 4000 Hz and 2600 Hz. The auditory system of H. crucifer is thus only roughly tuned to the temporal and spectral properties of the advertisement call. The female's specificity with respect to duration alone is adequate for species recognition, but intraspecific mate choice based on call frequency is extremely unlikely.     

Evidence for selective absorption of polyunsaturated fatty acids during digestion in the tobacco budworm, Heliothis virescens F.

Heliothis virescens F., the tobacco budworm, were reared individually on a semi-defined diet. The 5th and 6th instar larvae digested and absorbed approx. 72% of the available dietary lipid. Analysis of the fatty acid composition of total lipid in the diet and that remaining in the faeces indicated that the polyunsaturated fatty acids, linoleate and linolenate, were preferentially absorbed as compared to saturated and monoenoic fatty acids.     

Electrochemical investigations on several dimethylbenzo[a]pyrenes

Electrochemical studies on several dimethylbenzo[a]pyrene (DMBP)¹ derivatives were carried out at a platinum disk electrode employing cyclic voltammetric and chronopotentiometric techniques in (1:1) acetonitrile-benzene mixed solutions with tetra-n-butylammonium perchlorate as a supporting electrolyte. The results indicate that DMBPs undergo reversible one-electron oxidations and reductions to form the cation radical and the radical anion respectively as electrode products. The stability of the cation radicals of 1,3-, 2,3-, and 3,6-DMBPs is determined by the current reversal chronopotentiometric method in the medium studied at room temperature, and the pseudo-first-order decay rate constants of these radical species are 0.00046, 0.061, and 0.090 s^?1, respectively. Reactions of cation radicals with nucleophiles such as water and pyridine were also studied electrochemically and the result is described.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkaline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose-containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Regulation of oocyte maturation in the mouse: possible roles of intercellular communication, cAMP, and testosterone

Experiments were performed to determine if elevation of cumulus cell cAMP results in an increase in mouse oocyte cAMP while the heterologous gap junctions were intact. Both follicle-stimulating hormone (FSH) and cholera toxin induced a marked increase (>20-fold) in intracellular cAMP in isolated mouse cumulus cell-oocyte complexes in the presence of 3-isobutyl-1-methyl xanthine (IBMX). Concomitantly, both FSH and cholera toxin transiently inhibited resumption of meiosis of cumulus cell-enclosed but not denuded oocytes. The transient nature of the inhibitory effect produced by either FSH or cholera toxin was correlated with the cAMP level in the cumulus cell-oocyte complex. The inhibitory effect, however, was apparently not due to movement of cumulus cell cAMP to the oocyte via the functional heterologous gap junctions between cumulus cells and the oocyte. Radioimmunoassay of cAMP in oocytes free of attached cumulus cells or cumulus cell-enclosed oocytes exposed to either FSH or cholera toxin revealed that both groups of oocytes contained similar amounts of cAMP (about 0.14 fmole/oocyte). Metabolic labeling of cumulus cell-oocyte complexes with [³H]adenosine followed by incubation with either FSH or cholera toxin resulted in a marked increase in the amount of radiolabeled cAMP compared to that in unstimulated complexes. However, similar amounts of radiolabeled cAMP were found in oocytes derived from either stimulated or unstimulated complexes. Thus, we have not detected, using two methods of assay, that increasing the cAMP content of the cumulus cells results in any increase in the cAMP content of the oocyte. The apparent compartmentalization of cumulus cell cAMP elevated in response to either FSH or cholera toxin was not due to disruption of intercellular communication between the two cell types during the incubation; metabolic cooperativity was present between the two cell types and molecules of similar molecular weight and charge relative to that of cAMP were rapidly equilibrated between the two cell types. Testosterone potentiated the FSH/cholera toxin-induced transient inhibition of maturation of cumulus cell-enclosed oocytes. However, testosterone did not increase cAMP accumulation produced by either FSH or cholera toxin, decrease the rate of cAMP degradation, or promote movement of cumulus cell cAMP to the oocyte. Since cAMP elevated in response to FSH or cholera toxin appeared to be compartmentalized to cumulus cells and since neither FSH, cholera toxin, nor testosterone inhibited resumption of meiosis in denuded oocytes, it appears that the inhibitory effect promoted by FSH or cholera toxin is directly mediated by an agent other than cAMP, although cAMP generation is required for its action and that cumulus cells mediate the inhibition. These results are discussed in terms of a possible role of cAMP and steroids in regulating maturation in the mouse.     

Thermal inactivation of a Citrobacter ribonuclease: Influence of polyamines and ionic strength

The thermal inactivation of a Citrobacter sp. ribonuclease (RNase) is subject to control by a number of factors. Low concentrations of naturally occurring polyamines such as spermidine and spermine, and certain analogs of these compounds, protect the enzyme from inactivation. Changes in ionic strength cause wide variations in the rate at which enzyme activity is lost. Additionally, depending on the type of ion added to the reaction mixture, the rate constant for enzyme inactivation-may either increase or decrease as the ionic strength is raised. Thermodynamic parameters were determined under a variety of experimental conditions for the thermal inactivation of this RNase. It was found in all of these cases that the entropy of activation is large and negative, implying that a gross change in enzyme conformation is not taking place. The concentration and identity of ions present and the amount of polyamine available to interact with this RNase determines the rate of loss, by thermal inactivation, of enzyme activity in this in vitro system. These factors therefore constitute a system whereby substrate hydrolysis may be controlled with time.