Ovarian and fat body protein concentrations in Ips sexdentatus (Coleoptera: Scolytidae) parasitized by nematodes

The protein concentrations of fat body and ovaries in Ips sexdentatus either uninfected or infected by Parasitaphelenchus sp., P. sexdentati, or Contortylenchus diplogaster were measured at various stages of insect development, from preswarming maturation to the first oviposition (24 hr after mating). Weight variations of the fat body and ovaries in insects infected by C. diplogaster show the same evolution as those observed in uninfected insects, but at a much lower level. Fat body proteins in uninfected insects reach their minimum level during swarming, but they remain fairly constant throughout the maturation of the first egg. After dropping shortly after swarming, the ovarian protein level in such insects increases in two stages during ovarian maturation. The first stage, which corresponds to a slow protein incorporation, takes place during the first 12 hr after mating. During the second stage, i.e., beyond 12 hr, a significant level of proteins is rapidly incorporated into the ovaries. In insects infected by Parasitaphelenchus fat body proteins are reduced and protein incorporation into the ovaries is reduced; Parasitaphelenchus would thus affect at least some proteins required for ovarian maturation in their host. Fat body protein levels are even more affected by C. diplogaster than by Parasitaphelenchus, while incorporation into ovaries seems to be less affected in spite of slower ovarian growth. C. diplogaster might thus essentially act both upon proteins which are not required for the ovarian maturation of their host and upon nonproteinaceous substances that are required for such maturation. Results are discussed in relation to the possible mode of action of parasitic nematodes.     

Selective growth of sympathetic nerve fibers to explants of normally densely innervated autonomic effector organs in tissue culture

The synthesis of both total and electrophoretically fractionated proteins was studied in the wing epidermis of developing silkmoths, using a simple organ culture system which maintains the cells in synthetically normal condition for approximately 2 days. Differences were detected in the electrophoretic profile of proteins synthesized at different developmental stages, and correlated with transition from growth-related to differentiation-related products. Actinomycin D induced an apparently exponential decay in incorporation into total protein; the half-life of the decay ranged from 10 to 13 hr, depending on the stage of development. Double-label experiments indicated that decay of protein synthesis in the presence of actinomycin is not uniform, but proceeds at different rates for different components. Putative differentiation-specific products may be associated with differential resistance to actinomycin. At a specific developmental stage, actinomycin induced a transient secondary stimulation of overall incorporation. Measurements of leucine specific activity suggested that protein synthesis (rather than merely incorporation) is affected, and that “superinduction” is not limited to a few protein species.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Release of egg development neurosecretory hormone in Aedes aegypti and Aedes taeniorhynchus induced by an ovarian factor

Ovariectomized Aedes aegypti do not synthesize vitellogenin after a blood meal, unless an ovary from a blood-fed donor is implanted. Decapitation, however, prior to implantation inhibits vitellogenin synthesis. A female ovariectomized and decapitated 6 hr after a blood meal, synthesizes vitellogenin if an ovary from a blood-fed donor is implanted. On the other hand, females that are fed on blood and immediately decapitated can not be stimulated to synthesize vitellogenin with implanted ovaries removed from blood-fed donors. These experiments led to the hypothesis that the blood meal stimulates the ovary to secrete a corpus cardiacum stimulating factor, that in turn promotes release of egg development neurosecretory hormone stored in the corpus cardiacum.Injection of 20-hydroxy-ecdysone or ovarian extract prepared from ovaries removed from unfed females does not release egg development neurosecretory hormone. Thus corpus cardiacum stimulating factor is not 20-hydroxy-ecdysone, and ovaries removed from unfed females do not store it.The rate of inactivation of egg development neurosecretory hormone released from the corpus cardiacum after a blood meal was investigated by implanting an ovary into females that were blood fed for various intervals than decapitated and ovariectomized. Seventy per cent of implants grow when the operation is done 18 hr after feeding, and 30% when the operation is done between 18 and 24 hr after feeding, indicating that egg development neurosecretory hormone is stable for the first 18 hr after a blood meal.Aedes taeniorhynchus females ovariectomized 24 hr after adult emergence do not synthesize vitellogenin. When such a female is implanted with an ovary removed from a sugar-fed or blood-fed Aedes aegypti donor vitellogenin synthesis is initiated, and the implant grows. Decapitation prior to implantation inhibit vitellogenin synthesis and implants do not grow. These results indicate that corpus cardiacum stimulating factor is not species specific.     

The effects of starving versus fasting on blood composition in larval house crickets

Larval crickets (Acheta domesticus) starved for 2 days during the growth phase of the instar consumed twice as much water as larvae that ceased feeding of their own accord during the last 2 days of the last instar. The behaviour of drinking more water during starvation may compensate for dry weight loss and prevent the larvae from missing the critical weight required to initiate the next moult. During starvation the plasma volume increased while the tissue volume remained constant, which produced a shift in both organic and inorganic solutes from the tissues into the plasma. During fasting there was no change in tissue or plasma volume, therefore large osmotic adjustments were unnecessary, and the only change in plasma solutes noted was a decline in plasma proteins.The titres of proteins, lipids and amino acids remained constant during 2 days of starvation, though the amount of each increased because of the increased plasma volume. Although both the titre and the amount of plasma sugar sharply declined during starvation, there was no change in the sugar titre when the insects fasted. There was some evidence that prior to fasting the programmed gradual decline in food intake matched the decline in metabolic rate, which permitted a plasma sugar stability not evident in starved larvae. The decline in plasma proteins during the fasting phase appeared due to the removal of a larval specific protein and not a direct result of fasting.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

Effect of Triethyl Tin on Myelination in the Developing Rat

Myelinogenesis in developing rats was studied following chronic dosing with triethyl tin (TET), at a level of 1.0 mg TET/kg body wt/day. Experiments included starved controls with body weights depressed by 17 to 40% to equal those of the TET-treated groups. Rats at ages of 16, 21, and 30 days showed decreases relative to well-nourished controls in body weight, forebrain weight, myelin yield, cerebroside level, and specific activity of brain 2',3'-cyclic nucleotide-3'-phosphohydrolase when dosed with TET. At 30 days, myelin and cerebroside yields were reduced by approximately 55%, while CNP activity was reduced by less than 20%. No differences in the forebrain myelin protein composition between control, starved, and TET animals were noted. The rate of myelin protein synthesis relative to brain total protein (assayed by incorporation of intracranially injected [3H]glycine into brain homogenate and myelin proteins) was decreased in the TET rats in proportion to the decreased yield of myelin, but no particular myelin protein was preferentially affected. Matching starved controls exhibited similar body weight decreases, less pronounced forebrain weight decreases, and little or no decrease in myelin concentration. There was a relative increase in the myelin protein synthesis rate in the starved rats, indicating preferential utilization of limited protein precursors for myelin protein synthesis. Spinal cord myelin was also decreased in the TET rats, but less severely than in the forebrain. At all ages optic, but not sciatic, nerves showed decreases in myelin concentration with TET treatment. We conclude that TET inhibits forebrain growth and CNS myelination more severely than can be accounted for by a general metabolic insult.     

Biogenesis of mammalian mitochondria in the renoprival kidney. II. Aspects of mitochondrial DNA synthesis

Mitochondrial DNA (m-DNA) content and factors which might control its concentration were investigated in the renoprival kidney at various times after unilateral nephrectomy. On the basis of mitochondrial protein, m-DNA increased 30% in the renoprival kidney at 24 hr and returned to normal by 48 hr. The total tissue content of m-DNA was also increased at 24 hr. The specific activity of [³H]thymidine incorporated into m-DNA in vivo was decreased markedly at 24 hr after mononephrectomy; at the same time there was a threefold increase of [³H]thymidine incorporation into total cellular DNA. The incorporation into m-DNA was above normal at 48 hr. The mitochondrial specific DNase was decreased 60% at 24 and 36 hr post-mononephrectomy. There was no significant difference in the total radioactivity or total optical density at 260 nm of the acid soluble extract from mitochondria isolated at various times after mononephrectomy. The incorporation of [³H]thymidine into TMP and TDP in the renoprival kidney was not different from normal but there was a decrease in the incorporation into TTP. It is suggested that the increase in mitochondrial DNA could be due to a decrease in the rate of degradation rather than an increase in synthesis.     

Neurophysiological changes associated with paralysis arising from body stress in the cockroach, Periplaneta americana

Two hours after physical stress, Periplaneta americana could be separated into three behavioural categories: normal to hyperactive; torpid with ataxia; and paralysed. At 2 hr, 68 per cent were either torpid or paralysed, at 20 hr, 83 per cent were paralysed. Weight loss was a distinct physiological symptom of stress paralysis: The calculated mean loss was 9·5 mg/hr for torpid insects and 12·4 mg/hr for paralysed cockroaches, losses were four to six times larger than those occurring in starved cockroaches. However, the haemolymph osrnolarities of the three categories showed no appreciable differences. Only starved and paralysed cockroaches showed a noticeable reduction in muscle fibre membrane potentials of the flexor tibia—a mean value below 40 mV for starved insects and a mean value below 50 mV for paralysed insects. Both of these categories consistently showed a lower amplitude for junctional potentials, but paralysed cockroaches showed a much higher incidence of complete failure to neural stimulation. Most muscle fibres of completely paralysed insects lost their sensitivity to direct extracellular stimulation while the loss in sensitivity was less evident in starved cockroaches. Axonal conduction on the crural nerve was not changed by stress, and the spontaneous efferent activity of completely paralysed insects was similar to the pattern of activity for normal cockroaches. Stress seemed to alter the volume and content of the intermyofibral spaces of muscles.     

Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Synthesis of angiotensinogen by isolated rat liver cells and its regulation in comparison to serum albumin

Angiotensinogen (renin substrate) and albumin are synthesized by isolated hepatocytes almost linearly for 5 hr. The incorporation of radioactive leucine into total protein proceeded linearly for 3 hr. Without addition of amino acids to the incubation medium the synthesis of both proteins was still linear but fell off to 40% compared to the synthesis rate obtained by incubation with amino acids in serum concentrations. Higher amino acid concentrations could not further stimulate the synthesis. Addition or withdrawal of tryptophan had no effect on the synthesis rate of both proteins. After 5 hr incubation hydrocortisone had stimulated the incorporation of radioactive leucine into total protein by 13%, the albumin synthesis by 43%, and the angiotensinogen synthesis by 142%.     

Feeding causes the appearance of a factor in the haemolymph that stimulates protein synthesis

Soon after a locust (Locusta migratoria) begins to feed, an increase in protein synthesis can be detected in the animal. Isolation of fat body shows that this tissue synthesizes protein at a faster rate in recently fed animals than it does in fasting insects. Fasting locusts injected with haemolymph from fed insects increased protein synthesis when compared with locusts injected with haemolymph from fasting locusts. The factor causing this increase was present in the haemolymph within 5 min of feeding. Feeding or direct contact with the food was not essential to increase protein synthesis. Exposure of fasting locusts to feeding insects was sufficient to elevate the rates of protein synthesis in the fasting animals.The increase inprotein synthesis was not a result of general excitation or an increase in the concentration of tryptophan or isoleucine in the haemolymph. Ecdysteroid titres were uniformly low during the first ten days of adult life. Gel filtration of the fed haemolymph revealed a low molecular weight fraction (about 600 daltons) which stimulated protein synthesis upon injection into fasting locusts.     

Protein synthesis in ‘fat body’ and ovary of the physogastric queen of Macrotermes subhyalinus

In the physogastric queen of Macrotermes subhyalinus the fat body, when incubated in vitro with [¹⁴C] amino acids, synthesizes proteins at a much slower rate than ovarian tissue under the same conditions. Only a very small amount of the labelled proteins is released into the incubation medium. Oxygen consumption of the queen fat body is higher than that of ovarian tissue and the fat body of the king. At 3 hr after injection of [¹⁴C] amino acids in vivo the total fat body of the queen contains three to six times less labelled proteins than the two entire ovaries. It is assumed that in contrast to other insects the physogastric termite queen synthesizes vitellogenins mainly in the ovarian follicle cells and not in the fat body.The fat body of the king with a high incorporation rate of [¹⁴C] amino acids and a rapid release of synthesized protein into the incubation medium is comparable to the fat body of other insects.     

Juvenile hormone-controlled vitellogenin synthesis in Locusta migratoria fat body. Hormonal induction in vivo.

Biosynthetic processes related to the production of vitellogenin (yolk precursor protein) have been examined in the fat body of adult female Locusta migratoria. Vitellogenin-producing capacity was assayed by incubation of fat body with [³H]leucine, followed by precipitation from the medium with specific antiserum. In normal development, vitellogenin synthesis began at about Day 7 after emergence and became maximal at about Day 13, when this protein accounted for 60% of the total fat body protein output. The production of other proteins increased to a lesser extent, becoming maximal at about Day 6. The incorporation of uridine into fat body RNA rose to a maximum at Day 8, which coincided with a marked increase in tissue RNA content. The DNA content in adult female fat body approximately doubled between Days 3 and 8. Vitellogenin synthesis, and the increases in RNA and DNA, were prevented by removal of the corpora allata (the source of juvenile hormone). In allatectomized locusts, vitellogenin synthesis was induced by JH or an analog, ZR-515. Applied topically in acetone, these gave steep dose-response curves, half-maximal at 75 and 150 μg, respectively. After a single treatment with ZR-515, fat body vitellogenin production rose slowly during 48 hr, then steeply to a maximum at 72 hr, but after decay of this effect during 10 days, a second application of ZR-515 induced renewed synthesis with little initial lag. Hormone treatment produced a smaller increase in the output of other proteins, and an increase in incorporation into RNA which preceded the major rise in vitellogenin synthesis. Male fat body produced little or no vitellogenin. These results are consistent with action of JH at the gene level.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.