Anti-oxidative effects of silkworm storage protein 1 in HeLa cell

Silkworm hemolymph contains unique proteins that exhibit anti-apoptotic activity in mammalian cells. Among them, 30 K protein, which is one of the major anti-apoptotic molecules in silkworm hemolymph, has been well investigated. However, little is known about the biological functions of storage protein 1 (SP1), another main protein in silkworm hemolymph. In this study, the anti-apoptotic and anti-oxidative activities of SP1 were analyzed. A stable cell line expressing SP1 was constructed, which showed strong anti-apoptotic effect induced by staurosporine treatment. In addition, the cell line exhibited resistance to oxidative stress caused by hydrogen peroxide. For practical applications of SP1, recombinant SP1 was produced in Escherichia coli, and the supplementation of recombinant SP1 into culture medium exhibited anti-apoptotic and anti-oxidative activities. In addition, SP1 was found to be a cell-penetrating protein and localized in the cytosol as well as on the plasma membrane. The findings showed that SP1 itself is not an anti-oxidant; rather, it mediates intracellular anti-oxidative activity. In conclusion, the cellular resistance of SP1 to apoptosis and oxidative stress will provide a new strategy that could be utilized in the bio-industry for the production of biologics as well as for the development of anti-aging cosmetics.     

家蚕血液对BmE-SWU1细胞凋亡的抑制作用

以家蚕胚胎细胞系BmE-SWU1细胞为体外模型,用不同浓度的放线菌素D处理家蚕BmE-SWU1细胞进行家蚕细胞凋亡研究.结果表明:放线菌素D诱导家蚕细胞凋亡的作用呈时间、剂量依赖性.分别用浓度为0、50、100和200 ng/ml的放线菌素D处理BmE-SWU1细胞12 h后,凋亡峰所占比例分别为1.82%、1.26%、8.21%和12.31%.当放线菌素D的浓度为100 ng/ml时,诱导家蚕BmE-SWU1细胞凋亡的效果显著;家蚕血液对放线菌素D诱导的家蚕BmE-SWU1细胞凋亡具有明显的抑制作用.     

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.     

Deciphering the pathways of germ cell apoptosis in the testis

A growing body of evidence demonstrates that germ cell death both spontaneous (during normal spermatogenesis) and that induced by suppression of hormonal support or increased scrotal temperature occurs via apoptosis. The mechanisms by which these proapoptotic stimuli activate germ cell apoptosis are not well understood. In order to provide some insight, here we report the key molecular components of the effector pathways leading to caspase activation and increased germ cells apoptosis triggered by mildly increased scrotal temperature. Short-term exposure (43 °C for 15 min) of the testis to mild heat results, within 6 h, in stage- and cell-specific activation of germ cell apoptosis in rats. Initiation of apoptosis was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. Such relocation of Bax is further accompanied by sequestration of mitochondria and endoplasmic reticulum (ER) into paranuclear areas, cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and cleavage of PARP. Furthermore, Bax is co-localized with ER in the susceptible germ cells as assessed by combined two-photon and confocal microscopy and Western blot analyses of fractionated testicular lysates. In additional studies, using gld and lpr^cg mice, which harbor loss-of-function mutations in Fas-ligand (FasL) and Fas, respectively, we demonstrated that heat-induced germ cell apoptosis is not blocked, thus providing further evidence that the Fas signaling system is dispensable for heat-induced germ cell apoptosis in the testis. Taken together, these results demonstrate that the mitochondria- and possibly also ER-dependent pathways are the key apoptotic pathways for heat induced germ cell death in the testis.     

Oxidation-deficient silkworm hemolymph as a medium supplement for insect cell culture

Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wE^b showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wE^b showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wE^b hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wE^b hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.     

Inhibition of human cell apoptosis by silkworm hemolymph

Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture.     

Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines

A recombinant N-linked glycoprotein, secreted human placental alkaline phosphatase (SEAP), was produced in two Trichoplusia ni insect cell lines using the baculovirus expression vector. Silkworm hemolymph (SH) was added to TNMFH + 10% fetal bovine serum (FBS) medium to a concentration of 2.5% or 5%, and SEAP production and glycosylation in the presence of SH were compared with controls devoid of hemolymph. Growing Tn-4s cells in 5% SH-supplemented medium required progressive adaptation of the cells to SH, and adapted cells had a SEAP specific yield decreased by 2.5-fold compared with control cells not exposed to SH. Although SEAP produced in the control possessed little complex glycosylation (<1%), SEAP produced by SH-adapted cells in the presence of 5% SH possessed 8.7% sialylated structures, as well as unusual, asialylated, agalactosylated structures with a high degree of polymerization (DP). On the basis of enzymatic and mass-spectrometric analyses, we propose that these structures are glucosylated, high-mannose oligosaccharides. SEAP was also produced by Tn-4s cells without adaptation to SH when SH was added just prior to baculovirus infection, but SEAP specific yield was adversely affected (approximately fourfold reduction compared with control devoid of hemolymph), and glycosylation of SEAP produced under these conditions was characterized by large amounts of high-mannose and high-DP structures and an absence of complex structures. Similarly, Tn5B1-4 cells that were not adapted to SH had a SEAP specific yield reduced by approximately fivefold in SH-containing medium; however, these cells were able to produce 13.5% sialylated SEAP in the presence of 2.5% SH, whereas complex structures were not produced in the absence of SH. We propose that SH improves glycosylation either directly or indirectly by decreasing SEAP specific yield.     

Beneficial effect of silkworm hemolymph on a CHO cell system: Inhibition of apoptosis and increase of EPO production

To produce erythropoietin (EPO), Chinese hamster ovary (CHO) cells were first cultured in a medium containing FBS (growth medium) and then in a serum-free medium containing sodium butyrate (production medium). Sodium butyrate increases recombinant protein production, but also induces apoptosis, which reduces cell viability and productivity. In a previous study, we found that silkworm hemolymph (SH), an insect serum, inhibits the apoptosis of insect and mammalian cells. To overcome sodium butyrate-induced apoptosis, we added SH to growth medium. This pretreatment with SH inhibited the sodium butyrate-induced apoptosis of CHO cells and consequently increased their longevity and their ability to produce EPO. As a result, the volumetric productivity of EPO was increased five-fold. SH was found to inhibit cytochrome c release from mitochondria into the cytosol, and prevented the activation of caspase-3 and other subsequent caspase reactions.     

Death by design: mechanism and control of apoptosis

Active cellular suicide by apoptosis plays important roles in animal development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke and many neurodegenerative disorders. A central step in the execution of apoptosis is the activation of an unusual class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. Originally, the mechanisms for regulating the caspase-based cell death programme seemed to be different in Caenorhabditis elegans, mammals and insects. However, recent results suggest that these apparent differences in the control of cell death reflect our incomplete knowledge, rather than genuine mechanistic differences between different organisms.     

Death by design: mechanism and control of apoptosis

Active cellular suicide by apoptosis plays important roles in animal development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke and many neurodegenerative disorders. A central step in the execution of apoptosis is the activation of an unusual class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. Originally, the mechanisms for regulating the caspase-based cell death programme seemed to be different in Caenorhabditis elegans, mammals and insects. However, recent results suggest that these apparent differences in the control of cell death reflect our incomplete knowledge, rather than genuine mechanistic differences between different organisms.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.     

Death by design: mechanism and control of apoptosis

Active cellular suicide by apoptosis plays important roles in animal development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke and many neurodegenerative disorders. A central step in the execution of apoptosis is the activation of an unusual class of cysteine proteases, termed caspases, that are widely expressed as inactive zymogens. Originally, the mechanisms for regulating the caspase-based cell death programme seemed to be different in Caenorhabditis elegans, mammals and insects. However, recent results suggest that these apparent differences in the control of cell death reflect our incomplete knowledge, rather than genuine mechanistic differences between different organisms.     

造血器官机能障碍后家蚕血淋巴中蛋白质成分的变化

为了研究家蚕Bombyx mori造血器官机能障碍后其血淋巴中蛋白质成分的变化,利用重离子射线局部照射家蚕幼虫的造血器官,检测了照射后家蚕血淋巴中的蛋白质成分及注射大肠杆菌后在体内诱导出现的应急蛋白量的变化。结果表明,照射蚕血淋巴中的蛋白质含量与对照蚕之间没有明显的差异。但在成分分析时发现,5龄起蚕血淋巴中70 kD附近的3条蛋白质谱带比对照蚕的浓度要高,随着个体的发育两者的浓度都上升;5龄后期则相反,对照蚕的浓度比照射蚕高;脂肪体中贮藏蛋白质的含量具有相似的变化趋势。用家蚕贮藏蛋白质SP-1及SP-2的抗血清进行免疫印迹反应的结果显示：70 kD附近的3条蛋白质谱带的最上面的一条为贮藏蛋白质SP-1,下面的二条为贮藏蛋白质SP-2;同时照射蚕血淋巴中分子量约为24 kD的蛋白质成分也发生变化,5龄前期的浓度比对照蚕低,5龄第3天几乎检测不到;全体照射与造血器官局部照射蚕之间的结果相似。照射蚕注射大肠杆菌后在体内诱导出现的应急蛋白量明显比对照蚕要少。由此认为家蚕幼虫造血器官与血淋巴中的蛋白质成分有关,造血器官的机能障碍、血球的数量减少可影响脂肪体中蛋白质的合成,从而使存在于血淋巴中的蛋白质成分发生变化。     

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.     

Aspirin induces apoptosis through mitochondrial cytochrome c release

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VAD·fmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release.     

Yeast as a tool to study Bax/mitochondrial interactions in cell death

The budding yeast Saccharomyces cerevisiae has proven to be a powerful tool in investigations of the molecular aspects of the events involved in apoptosis, particularly the steps implicating mitochondria. Yeast does not have obvious homologs of the proteins involved in the regulation of apoptosis, and provides a simplified model system in which the function of these proteins can be unraveled. This review focuses on the interactions of two of the major pro-apoptotic Bcl-2 family members, Bax and Bid, with mitochondria. It is shown that yeast has allowed questioning of several crucial aspects of the function of these two proteins, namely the molecular mechanisms driving their insertion into the mitochondrial outer membrane and those leading to the permeabilization to cytochrome c. More recently, signaling pathways leading to Bax-induced cell death, as well as other forms of cell death, have been identified in yeast. Both 'apoptosis-like' and autophagy-related forms of cell degradation are involved, and mitochondria play a central role in these two signaling pathways.     

Identification of a novel hemolymph peptide that modulates silkworm feeding motivation

Phytophagous insects do not constantly chew their diets; most of their time is spent in a non-feeding quiescent state even though they live on or around their diets. Following starvation, phytophagous insect larvae exhibit enhanced foraging behaviors such as nibbling and walking similar to the sequential behavior that occurs prior to each meal. Although extensive physiological studies have revealed regularly occurring feeding behaviors in phytophagous insects, little has been elucidated regarding the mechanism at the molecular level. Here, we report identification and characterization of a novel 62-amino acid peptide, designated as hemolymph major anionic peptide (HemaP), from the hemolymph of Bombyx mori larvae that induces foraging behaviors. The endogenous HemaP levels are significantly increased by diet deprivation, whereas refeeding after starvation returns them to basal levels. In larvae fed ad libitum, hemolymph HemaP levels fluctuate according to the feeding cycle, indicating that locomotor-associated feeding behaviors of B. mori larvae are initiated when HemaP levels exceed an unidentified threshold. Furthermore, administration of exogenous HemaP mimics the starvation-experienced state by affecting dopamine levels in the suboesophageal ganglion, which coordinates neck and mandible movements. These data strongly suggest that fluctuation of hemolymph HemaP levels modulates the regularly occurring feeding-motivated behavior in B. mori by triggering feeding initiation.     

Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori

Abstract Two‐dimensional gel electrophoresis (2‐DE) followed by matrix‐assisted laser desorption ionization – time‐of‐flight/time‐of‐flight mass spectrometry (MS) analysis were used to charaterize the hemolymph proteomic profiles of the silkworm, Bombyx mori. At days 4 (V4) and 5 (V5) of the fifth (final) instar, when the larvae were at the fast‐growing stage, we found dramatic changes in spots representing proteins having an approximate molecular weight (MW) of 30 kDa. Of these spots, four 30K proteins were highly up‐regulated, implying a close association with the growth and development of B. mori larvae. To understand the molecular basis and underlying mechanisms involved in development and metamorphosis, the proteome of whole hemolymph at V5 was analyzed using shotgun liquid chromatography tandem mass spectrometry with an LTQ‐Orbitrap. A total of 108 proteins were identified without any false discovery hits. These proteins were involved in a variety of cellular functions, including metabolism, development, nutrient transport and reserve, and defense response. Gene ontology analysis showed that 3.4% of these proteins had nutrient reservoir activities and 5.7% were involved in the response to stimulus. Pathway analysis revealed that 22 proteins with common targets were involved in various cellular processes such as immunity, differentiation, proliferation and metamorphosis. These results suggested that some key factors such as the 30K proteins in hemolymph play important roles in B. mori growth and development. Moreover, the multiple functions of hemolymph may be operated by a complex biological network.     

Heme deficiency causes apoptosis but does not increase ROS generation in HeLa cells

Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.