Enhancement of sialyltransferase-catalyzed transfer of sialic acid onto glycoprotein oligosaccharides using silkworm hemolymph and its 30K protein

Silkworm hemolymph (SH) has been reported to inhibit apoptosis in both insect and human cells, and increase the high-sialylation structure of recombinant glycoprotein in insect cells. This indicates that SH might increase glycosyltransferase activity. Therefore, this study examined the effect of SH on the activity of sialyltransferase, which catalyzes the sialylation of the glycoprotein. When 10 μg/mL of SH was added to the reaction mixture, almost complete sialylation was observed even under the reaction conditions where sialyltransferase-catalyzed sialylation rarely occurs. The effect of deproteinized SH (dSH) and the 30K protein, which is a major plasma protein in SH, was examined to determine which component in SH enhances sialylation. The 30K protein promoted sialylation, while the dSH did not. This suggests that SH and its 30K protein can be used as an additive to a medium for efficient glycosylation when mammalian cells are being cultured for the production of valuable biopharmaceuticals, many of which are glycoproteins.     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis

Silkworm hemolymph (SH) was found to exhibit anti-apoptotic activities in mammalian and insect cell systems. An anti-apoptotic mechanism of SH was investigated in a staurosporine-induced HeLa cell using flow cytometry, caspase assay, Immunoblot, and Immunochemistry. The addition of 5% SH to the medium resulted in lower intracellular activities of caspase-3 and caspase-9 after 0.6 μM of staurosporine treatment; however, SH did not directly inhibit the activities of those enzymes. This suggests SH inhibits the event upstream of these caspase activation steps, such as mitochondrial level events. We found from Immunoblot and Immunochemistry that cytochrome c release from the mitochondria was blocked by SH. SH also inhibited Bax translocation to the mitochondria. On the contrary, SH did not block the apoptosis when Bax is not involved in promoting apoptosis. With these results, we propose that SH protects mitochondria from apoptosis signal via blocking Bax translocation, and the subsequent apoptotic events are then inhibited. The inhibition of apoptosis using SH and its components may lead to new approaches for the minimization of cell death during commercial animal cell cultures.     

淡足侧沟茧蜂寄生对甜菜夜蛾幼虫血淋巴总糖、蛋白质及虫体脂质含量的影响

为探讨淡足侧沟茧蜂Microplitis pallidipes Szepligeti调控寄主的生理机制,测定了淡足侧沟茧蜂寄生后,甜菜夜蛾Spodoptera exigua(Hübner)幼虫血淋巴总糖、蛋白质及虫体脂类含量的变化。研究结果表明,寄生后连续5 d的观察时间内,甜菜夜蛾幼虫血淋巴总糖含量从寄生后第1天开始就高于未寄生寄主幼虫,且在寄生后第2至5天达到显著水平;除寄生后第3天外,被寄生寄主幼虫血淋巴总蛋白质含量始终低于未寄生幼虫,且在寄生后第1、4、5天达到显著水平;甜菜夜蛾被寄生后,虫体脂质含量始终高于未寄生幼虫,且从寄生后第2天开始达到显著水平。本研究揭示,淡足侧沟茧蜂寄生刺激了寄主甜菜夜蛾幼虫血淋巴总糖合成,但抑制了其蛋白质合成,同时也刺激了甜菜夜蛾虫体脂质合成。     

昆虫抗菌肽研究和应用现状

昆虫在受到刺激或感染之后,在其血淋巴中会产生一种抗菌类物质,称抗菌肽。抗菌肽具有分子小、稳定性好、广谱抗菌、无毒副作用等特点,在农业、医药、食品等领域有广泛的应用前景。简要综述了昆虫抗菌肽的基础研究和应用现状。     

Impact of metal accumulation pattern on the annual rhythmicity of antioxidants and their interrelationship to maintain the oxidative balance in mollusc

A chronological relationship between the annual profiles of oxidative stress markers, the key regulator of stress physiology has been sought in a terrestrial mollusc (Nerita articulata) under natural photothermal conditions. The hemolymph samples were collected at two different times in each month (from January to December) and the same was repeated for two consecutive years throughout an annual cycle. The fluctuations in the concentrations of certain heavy and trace metals (zinc, copper, cadmium, mercury, lead, and nickel) in both soil and hemolymph of Nerita are also estimated accordingly. Therefore, the present study aims to explore the rhythmic responses of oxidative stress marker to assess the impact of different trace and heavy metals on selected mollusc species. We tries to develop a realistic conceptual idea to analyze and predict the effect of changing environmental pollution on the possible shift in the rhythmicity of aforesaid antioxidants in terrestrial mollusc and their adaptive responses to thrive in such environment. Our results indicates that the amplitude of circannual rhythms of all the selected stress markers varied accordingly but the pattern of annual fluctuation is noted to be similar, and correlated with the metal accumulation. Therefore current information might help to frame the adaptive strategies for invertebrate species under similar toxic circumstances.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

Photoperiod Is the Main Cue That Triggers Supercooling Ability in the Land Snail, Helix aspersa (Gastropoda: Helicidae)

Helix aspersa hibernates in Brittany (western France), where it may experience subzero temperatures. Studies on cold hardiness, although scarce in land snails, have shown a seasonal variation in supercooling ability, associated with high temperatures of crystallization (Tc). In the present work, two key environmental factors, temperature and photoperiod, were studied to elucidate, how they may affect the enhancement of supercooling ability in the snails from the end of summer to winter. Nine groups of adult snails were acclimated to different combinations of photoperiod (LD-16:8, LD-12:12, and LD-8:16 h) and temperature (15, 10, and 5°C). Temperature of crystallization, hemolymph osmolality, and water content were measured. The results demonstrate a significant effect of the photoperiod on Tc, i.e., shorter photoperiods induce lower Tc (LD-16:8 h, mean Tc = −3.0°C, SD = 2.0; LD-12:12 h, mean Tc = −4.3°C, SD = 1.9; LD-8:16 h, mean Tc = −5.2°C, SD = 1.9; n = 90), whereas the acclimation temperature had no effect. Measurements of hemolymph osmolality and water content showed that osmolality is negatively correlated with water content. Mechanisms such as dehydration are involved in the decrease of Tc. A declining photoperiod triggers a lower Tc, long before the onset of winter conditions. This response may have an adaptive component, allowing individuals to cope with the mild winters typically observed in oceanic regions.     

Nonvitellogenic female protein in Musca domestica

During vitellogenesis in Musca domestica, a major hemolymph protein, in addition to vitellogenin, appears preferentially in females. This protein is synthesized by the adult fat bodies, secreted into the hemolymph, and is not taken up by the ovaries during vitellogenesis. We have designated this protein nonvitellogenic female protein (NVFP). It is composed of only one type of polypeptide (M_r=70,000) and occurs in two different forms. Synthesis of NVFP is induced by a protein diet, attaining maximum concentrations in females at the middle of the gonotrophic cycle. In males its maximum concentration never surpasses 10% of the concentration in females. The quantitative variation of the NVFP is cyclic and coincident with the gonotrophic cycles of Musca domestica.     

Properties,synthesis, and accumulation of storage proteins in Pieris rapae L.

Two kinds of storage proteins (SP-1, SP-2) were confirmed in hemolymph and fat body of Pieris rapae during metamorphosis. Both proteins were present in high concentrations in the hemolymph during the last larval instar. Hemolymph concentrations of SP-1 and SP-2 dropped after pupation as the proteins were being deposited in fat bodies. SP-2 is present in a larger amount than SP-1. Detailed studies on storage proteins determined their properties, mode of synthesis, and accumulation in the fat body. SP-1 has a molecular weight of 500,000 and consists of one type of subunit (M_r 77,000), while SP-2 has a molecular weight of 460,000 and is composed of two types of subunits (M_r 80,000 and 69,000). The pl values of SP-1 and SP-2 were determined to be 6.97 and 7.06, respectively. Fat body cells from 1-day-old fifth instar larvae synthesized storage proteins in large amounts, whereas those from late prepupae exhibited high protein sequestration. Proteins taken up in fat body accumulated in dense granules during the pupal stage but sharply decreased at the adult stage. Morphological changes in the fat body tissues were observed during the larval-pupal transformation; the nuclei of fat body cells became irregularly shaped, and the boundaries between cells seemed to be obscure. Synthesis, storage, or degradation of storage proteins in fat body during development is closely associated with morphological changes in the tissues.