Binding of insecticides to lipophorin and arylphorin,two hemolymph proteins of Heliothis zea

The binding of different insecticides to hemolymph proteins of the corn earworm, Heliothis zea, was studied by an analytical isoelectric focusing technique. Two proteins capable of binding hydrophobic substrates were discovered and identified as lipophorin and arylphorin. Lipophorin, the major lipoprotein of Heliothis zea, demonstrated rather unspecific binding of xenobiotics of different hydrophobicities. Arylphorin, a putative storage protein, revealed strong affinity for compounds of medium polarity, binding only weakly to insecticides of higher or lower polarity.     

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers.     

Growth factors in invertebrate in vitro culture

Summary An increasing number of polypeptide growth factors have been identified that have proven essential in the development of defined cell culture media for mammalian cell culture. The development of defined mammalian cell culture media, in turn, has provided an environment for studying cell lines in an experimentally manageable unit for studying the action of cellular regulators and genes that determine the properties of cells. Evidence that vertebrate growth factors may be present in insects is based on DNA sequences that encode epidermal growth factor and transforming growth factor-β. However, research on the influence of commercially available vertebrate growth factors is very limited. Although the majority of insect growth-promoting substances studied were isolated directly from insect hemolymph, few of these have been purified to the extent that they could be tested in insect cell, tissue, and endoparasite cultures. Research is needed in both of these areas to aid in developing defined insect culture systems, and to understand better the regulation of postembryonic growth and development in insects.     

Streptococcal immunoglobulin-binding protein Sib35 exerts stimulatory and mitogenic effects toward mouse B lymphocytes

Group A Streptococcus (GAS) produces several immunopotent proteins such as the immunoglobulin-binding protein 35 (Sib35) family, known to be an immunoglobulin G-degrading enzyme of GAS and a CD16-binding protein of GAS, Mac-1-like protein/IdeS. In this study, Sib35 activated mouse B cells and induced the proliferation of B lymphocytes, while it also bound directly to B cells, which enhanced the expression of MHC class II and B7-2. Furthermore, Sib35 induced the differentiation of B cells to immunoglobulin-producing plasma cells and B cell line proliferation. These results suggest that Sib35 functions as a streptococcal mitogen of mouse B cells.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

In vitro properties of various growth factors and in vivo effects

This article summarizes some of the data that have been accumulated on several growth factors. Biochemical and biological properties of the Epidermal, Fibroblast, Astrocytes and Tumor growth factors (EGF, FGF, AGF, TGF) and those of growth factors derived from Platelets (PDGF), Brain (BDGF, ECGF), Eye (EDGF) and Cartilage (CDGF) are reviewed, as well as the in vitro mechanism of action of EGF and PDGF. The in vivo effects of these growth factors, particularly the experiments achieved to understand the physiological or physiopathological significance are described. The potential interest of these molecules in pharmacology and their use as wound healing agents is discussed.     

Rainbow trout (Oncorhynchus mykiss) sIgM- leucocytes secrete an interleukin-2 like growth factor after mitogenic stimulation in vitro

Two secreted proteins were detected in culture supernatants of PHA or PMA stimulated, immunomagnetically separated, sIgM(-) leucocytes of rainbow trout (Oncorhynchus mykiss) with 60kDa and with 12-15kDa (multiple bands). So called conditioned media (CM), containing these proteins, induced significant activation of blood and head kidney leucocytes. Immunomagnetically separated, naive as well as PHA activated sIgM(-) T lymphocytes and LPS prestimulated sIgM(+) B lymphocytes could be identified to be responding to these secreted proteins. Using a monoclonal antibody specific for mouse IL-2 (clone JES6-1A12), one of the multiple 12-15kDa proteins could be stained in Western blots. It was also shown that the induced proliferation was due to this protein in the CM, as the same anti-IL-2 mab was able to block the CM induced proliferation. Furthermore, survival of the IL-2 dependent mouse cell line HT-2 was enhanced after addition of various concentrations of CM. The data presented show, for the first time, that mitogen stimulated trout sIgM(-) leucocytes secrete a cytokine like growth factor sharing functional and structural similarities with mammalian IL-2.     

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.     

Growth and development of tomato fruitsin vivo andin vitro

Tomato (Lycopersicon esculentum Mill.) fruits of the male sterile cultivar Pearson (MS35 BC4, 61) were transferred toin vitro culture during the cell division period. Fruits grownin vivo andin vitro were compared throughout their development according to various growth parameters: fresh and dry weight, cell number, cell diameter, and DNA and total protein content. In all cases, the values pertaining to fruits grownin vitro were significantly lower than theirin vivo counterparts. The final fresh weight of fruits transferred to culture 2, 5, or 10 days after pollination was only 0.7, 1.2, and 3.4%, respectively, of that of plant-grown fruits. The results indicate that the reduced fruit sizein vitro is related to the reduction in both cell number and cell size. It is interesting to note that the DNA content per cell increased 15-fold during the growth of the plant-grown fruits while this accumulation was only between 2-and 3-fold in all the cultured fruits. The time to first colour appearance of fruits cultured 2, 5, or 10 days after pollination was 196, 132 and 85%, respectively, of that of plant-grown fruits.     

Anti-allergic effects of His-Ala-Gln tripeptide in vitro and in vivo

We examined the inhibitory effects of HAQ (His-Ala-Gln) peptide on type-1 allergy in vitro and in vivo. HAQ peptide inhibited β-hexosaminidase release and intracellular Ca²⁺ levels of rat basophilic leukemia RBL-2H3 cells. Oral administration of a HAQ peptide-added diet (1 mg/mouse/administration) to C3H/HeJ mice for 14 days led to significant suppression of allergic symptoms, but did not reduce allergen-specific IgE or IgG₁.     

Cytotoxic effects of nanoparticles assessed in vitro and in vivo

An increasing number of applications is being developed for the use of nanoparticles in various fields. We investigated possible toxicities of nanoparticles in cell culture and in mice. Nanoparticles tested were Zn (300 nm), Fe (100 nm), and Si (10-20, 40-50, and 90-110 nm). The cell lines used were brain, liver, stomach, and lung from humans. In the presence of nanopaticles, mitochodrial activity decreased zero to 15%. DNA contents decreased zero to 20%, and glutathione production increased zero to 15%. None of them showed a dose dependency. Plasma membrane permeability was not altered by nanoparticles. In the case of Si, different sizes of the nanoparticles did not affect cytotoxicity. The cytotoxicity was also shown to be similar in the presence of micro-sized (45 microm) Si particles. Organs from mice fed with nanoparticles showed nonspecific hemorrhage, lymphocytic infiltration, and medullary congestion. A treatment with the micro-sized particle showed similar results, suggesting that the acute in vivo toxicity was not altered by nano-sized particles.     

Insulin-like Growth Factor-I cDNA Gene Transfer in vitro and in vivo

Our hypothesis is that gene transfer of an IGF-I CMV-cDNA with cholesterol containing cationic liposomes is an efficient tool for transient transfection of growth factors in vitro and in vivo. In vitro, we transiently cotransfected IGF-I cDNA with a CMV construct and a Lac Z CHKCHK-galactosidase cDNA/CMV construct using cholesterol containing cationic liposomes and measured CHKCHK-galactosidase and IGF-I mRNA and protein. In vivo, we subcutaneously injected 3-month-old male Sprague–Dawley rats with IGF-I cDNA and CHKCHK-galactosidase cDNA into rat skin. After IGF-I and CHKCHK-galactosidase were cotransfected into PC12 cells, Northern blot analysis showed that the peak time of IGF-I expression was 2 days for mRNA and 5 days for protein. In vivo, a cDNA/liposome ratio of 1:2 was most effective. IGF-I protein expression in IGF-I-transfected skin resulted in significant transfection from day 5 to day 7. In situ determination of CHKCHK-galactosidase activity confirmed that transfections resulted in a restricted expression area.     

Hormonal effects on insects and other endoparasites in vitro

Summary Metamorphic and reproductive events in vertebrates and invertebrates are under endocrine control and are often correlated with developmental, behavioral, or reproductive changes in the parasites living in or on these hosts. This paper reviews selected examples ofa) host hormone mediated influences on endoparasites in vivo,b) host hormone effects in vitro on protozoan, helminth, and insect endoparasites, andc) identifies possible relationships in hormone effects across parasite taxa. The significance of studies on endoparasites in vitro in relation to the impact of host hormones, antihelminthic, and prophylactic drugs on parasite growth and proliferation will also be addressed. A review of the literature indicates only limited studies have been done in vitro in an attempt to elucidate the bases of reported host hormone influences on endoparasites in vivo. Steroid hormones of hosts seem to stimulate growth, molting or encystment or both of helminth, insect, and protozoan parasites. Vertebrate steroids such as estrogen, testosterone, and progesterone had primarily reproduction- or growth-promoting effects or both on protozoan and nematode parasites. Insect ecdysteroids such as ecdysone, 20-hydroxyecdysone, and makisterone were the most widely studied steroids in vitro and induced growth or molting or both of cestode, nematode, and insect parasite larvae. Although juvenile hormone (JH III) stimulated growth in the protozoan and nematode parasites tested, the analogue methoprene and JH precursors, farnesal, farnesol, and farnesol methyl ether had various effects. Biogenic amines also varied in their effects on the nematode parasites tested, while the peptide hormone, insulin, stimulated growth in the protozoans tested. The evidence for in vitro effects of host hormones on their natural endoparasites is patchy at best. Additional studies are needed to identify the biochemical bases for the numerous host hormone mediated effects on parasites. This work was supported by grant DCB 8502235 from the National Science Foundation, Washington, DC, GAM 8700433 from the United States Department of Agriculture, Washington, DC. This work was supported by grant DCB 8502235 from the National Science Foundation, Washington, DC, GAM 8700433 from the United States Department of Agriculture, Washington, DC.     

Comutagenic and coclastogenic effects of selenium in vitro and in vivo

The comutagenic activity of selenium was investigated using in vitro and in vivo techniques, including the liquid suspension modification of the standard Salmonella/microsome mutagenicity assay, the metaphase analysis of chromosome aberrations in CHO cells and in mouse bone marrow as well as the micronucleus assay in mouse bone marrow. 4 h growth of S. typhimurium TA1535 in a nutrient broth containing 2.9 x 10(-5) M but not 1.16 x 10(-5) M Na2SeO3 caused an up to 10-fold increase of the number of N-methylnitrosourea (MNU, 2.0-2.5 mM)-induced his+ revertants and an up to 2-fold elevation of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1.48 x 10(-5))-induced mutation rate. Pretreatment of bacteria with Na2SeO3 alone had no effect on the spontaneous mutation level. The combined treatment of CHO cells with MNNG (1.25 x 10(-5) M) or tobacco smoke (TS, 2-3 puffs generated by a cigarette inhalation machine) plus Na2SeO3 (0.58-1.16 x 10(-5) M) starting 2 h and 4 h before the MNNG or TS treatment respectively resulted in a 2-3-fold increase in the percent of metaphases with chromosome aberrations. Furthermore, treatment for 7-14 days of male BDF1 (C57Bl x DBA2) or CC57W mice with Na2SeO3, added to the drinking water at a concentration of 10 ppm, potentiated by 2-3 times the chromosome-damaging activity of urethane (0.5-1.0 g/kg, i.p.) in mouse bone marrow, as measured by the formation of micronuclei or chromosome aberrations. In addition, Na2SeO3 increased up to 43.8% the number of micronucleated polychromatic erythrocytes (MNPCE) induced by mitomycin C (MMC, 1.5 mg/kg, i.p.) in BDF1 mouse bone marrow. Treatment of mice with Na2SeO3 alone had no effect on the spontaneous level of MNPCE. All these findings are consistent with a comutagenic and coclastogenic activity of selenium both in prokaryotes and in eukaryotes, in vitro as well as in vivo after pretreatment of target cells with the trace element.