Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid

A novel process is presented for the isoelectric precipitation of soy protein, using carbon dioxide as a volatile acid. By contacting a soy meal extract with pressurized carbon dioxide, the solution pH was decreased to the isoelectric region of the soy proteins. Complete precipitation of the precipitable soy proteins could be achieved for protein concentrations up to 40 g/l at pressures less than 50 bar. Isoelectric precipitation with a volatile acid enabled accurate control of the solution pH by pressure and eliminated the local pH overshoot, usual in conventional precipitation techniques. The advantage of the improved precipitation control was reflected by the morphology of the precipitate particles. Protein aggregates formed by CO₂ were perfectly spherical whereas protein precipitated by sulfuric acid had an irregular morphology. The influence of process variables to control particle size is discussed.     

家蚕耐氟性差异的细胞化学研究

对不同蚕品种的耐氟性、ACPase的氟敏感性、蚕品种耐氟性机理的研究表明,在供试蚕品种中以浙农1号的耐氟性最强,杭 8的耐氟性最弱;家蚕Bombyx mori血淋巴ACPase活性与蚕品种的耐氟性无明显关系;氟对蚕的血淋巴和中肠组织细胞的ACPase活性都有抑制作用,并随着氟添食浓度的增加ACPase活性降低,但超过一定浓度的氟添食,血淋巴ACPase活性反而有一个回升的过程,这个转折点出现可能的浓度及回升的幅度与蚕品种的耐氟性有关;细胞化学研究发现此转折点的出现是由于高浓度氟引起细胞结构的破坏而导致蚕体组织细胞内的ACPase大量向血腔释放的结果;氟敏感性蚕品种杭 8在很低氟量添食即可引起中肠组织细胞的ACPase大量向血腔释放,使血淋巴中的ACPase活性大幅度上升,随后ACPase活性受到完全的抑制;耐氟性较强的蚕品种浙农1号则在较高的氟含量添食时才向血腔释放ACPase,且血淋巴中ACPase增高的幅度小,在很高的氟量添食时全面抑制中肠ACPase活性。氟对不同品种ACPase活性影响的差异被认为是家蚕品种耐氟性差异机理之一。     

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.     

Control of the circadian rhythm of carbon dioxide assimilation in Bryophyllum leaves by exposure to darkness and high carbon dioxide concentrations

The circadian rhythm of CO₂ assimilation in detached leaves of Bryophyllum fedtschenkoi at 15° C in normal air and continuous illumination is inhibited both by exposure to darkness, and to an atmosphere enriched with 5% CO₂. During such exposures substantial fixation of CO₂ takes place, and the malate concentration in the cell sap increases from about 20 mM to a constant value of 40–50 mM after 16 h. On transferring the darkened leaves to light, and those exposed to 5% CO₂ to normal air, a circadian rhythm of CO₂ assimilation begins again. The phase of this rhythm is determined by the time the transfer is made since the first peak occurs about 24 h afterwards. This finding indicates that the circadian oscillator is driven to, and held at, an identical, fixed phase point in its cycle after 16 h exposure to darkness or to 5% CO₂, and it is from this phase point that oscillation begins after the inhibiting condition is removed. This fixed phase point is characterised by the leaves having acquired a high malate content. The rhythm therefore begins with a period of malate decarboxylation which lasts for about 8 h, during which time the malate content of the leaf cells must be reduced to a value that allows phosphoenolpyruvate carboxylase to become active. Inhibition of the rhythm in darkness, and on exposure to 5% CO₂ in continuous illumination, appears to be due to the presence of a high concentration of CO₂ within the leaf inhibiting malic enzyme which leads to the accumulation of high concentrations of malate in the leaf cells. The malate then allosterically inhibits phosphoenolpyruvate carboxylase upon which the rhythm depends. The results give support to the view that malate synthesis and breakdown form an integral part of the circadian oscillator in this tissue.Abbreviations B. Bryophyllum - PEPCase phosphoenolpyruvate carboxylase     

Studies on the Sterol of Bombyx mori L

In whole stages of Bombyx silkworm it was shown with gas-liquid chromatographic systems that silkworm-sterols consist of three sterols such as cholesterol, β-sitosterol, and campesterol, at least, and the sterols in the 5th instar larva contain afore-mentioned three sterols and an additional unknown sterol. Moreover, the sterol composition of the silkworm was studied in various stages.     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.     

Effect of carbon dioxide on yeast growth and fermentation

Inhibition of yeast function by ethanol and by high substrate concentrations is well recognized and, to a limited extent, quantified. The role of carbon dioxide in affecting yeast metabolism (particularly growth processes) is not clear although inhibition is generally found at moderate to high concentrations of the dissolved gas. A similar situation exists with other microorganisms and with other fermentation systems. An understanding of the role of carbon dioxide, and particularly of its inhibitory effects on enzyme action and membrane function, is required if the observed global inhibition of yeasts and other fermentation systems is to be partitioned to its appropriate causes.     

N-linked glycan structures of mouse interferon-beta produced by Bombyx mori larvae

The full-length mouse interferon-beta (mIFN-beta) cDNA, including the secretion signal peptide coding region under control of the polyhedrin promoter, was introduced into Bombyx mori nucleopolyhedrovirus (BmNPV). Recombinant mIFN-beta (rmIFN-beta) was accumulated in the haemolymph of infected silkworm larvae. Western blot analysis showed isoforms of rmIFN-beta, suggesting that rmIFN-beta is glycosylated. The glycan structures of purified rmIFN-beta were determined. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by gel filtration, reversed-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains were identified by a combination of two-dimensional PA-sugar chain mapping, MS analysis, and exoglycosidase digestions.     

Metal-free carbon dioxide reduction and acidic C-H activations using a frustrated Lewis pair

Activation of CO₂ and acidic C-H bonds by the lutidine-tris(pentafluorophenyl)borane [Lut/B(C₆F₅)₃] frustrated Lewis pair (FLP) are described (lutidine = 2,6-dimethylpyridine). Lut/B(C₆F₅)₃ reacts with CO₂ and H₂ at ambient temperature and 4 atm of pressure to form the lutidinium boro-formate salt [LutH⁺][HC(O)OB(C₆F₅)₃⁻]. This salt has been fully characterized including an X-ray crystal structure and independent synthesis from formic acid and Lut/B(C₆F₅)₃. Attempts to activate a C-H bond in methane by Lut/B(C₆F₅)₃, analogous to its heterolytic cleavage of H₂, were unsuccessful, which are consistent with published calculations showing significant barriers to this reaction. Lut/B(C₆F₅)₃ does react with more acidic C-H bonds, including acetone and nitroalkanes. With nitromethane, the boro-nitrone anion H₂CNO₂B(C₆F₅)₃⁻ is formed, as indicated by NMR and mass spectral analyses.     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Sterol composition in larvae of the silkworm, Bombyx mori

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

Studies on the utilization of single cell protein by the silkworm Bombyx mori

Final-instar larvae of Bombyx mori fed mulberry leaves, supplemented with Spirulina fusiformis (Woronichin) as a source of single cell protein (SCP), required 6 days to attain a maximum larval weight of 2090 mg; control group larvae needed 9 days to attain a final larval weight of 1470 mg. Quantity of feeding, assimilation and conversion efficiencies increased substantially in the SCP-fed group. Significant improvements in the economic characters such as cocoon, pupal, and shell weights were obtained in the SCP supplemented larvae in comparison to the normal leaf fed larvae. About 15% of the labelled S. fusiformis was directly incorporated into larval tissue. Presence of SCP in the gut facilitated better conversion of consumed leaf protein.

---

Etudes sur l'utilisation des protéines de cellules isolées par le ver à soie, Bombyx mori

Résumé Des chenilles du dernier stade de Bombyx mori, alimentées sur mûrier additionné de Spirulina fusiformis comme source de protéine de cellule isolée (SCP), atteignent en 6 jours le poids larvaire maximum de 2090 mg; les chenilles témoins consommaient pendant 9 jours pour obtenir leur poids larvaire final de 1470 mg. Les quantités consommées, les coefficients d'assimilation et de conversion ont augmenté substantiellement chez les chenilles avec SCP. Des augmentations significatives de critères économiques, comme les poids de cocon, de nymphe et de cogul, ont été observées avec l'addition de SCP par rapport aux témoins. Environ 15% du S. fusiformis marqué a été incorporé directement dans les tissus larvaires. La présence de SCP dans l'intestin a permis une meilleure conversion des protéines foliaires consommées.

     

Fluxes of carbon dioxide and water vapor above paddy fields

Atmospheric fluxes of carbon dioxide and water vapor were measured by the eddy correlation technique over a paddy field in 1989. The carbon dioxide was transported downward during daylight hours due to photosynthesis of the paddy crop. The downward flux of carbon dioxide increased with increasing net radiation. Maximum values of downward flux varied with the growing stage of the paddy crop: ca. 0.3 mg m^–2 s^–1 at early vegetative growth stage and ca. 1.3 mg m^–2 s^–1 at ear formation stage. The daytime totals of downward flux of carbon dioxide also showed seasonal variation reflecting the photosynthetic activity of the paddy crop: ca. 6 g m^–2 at early vegetative growth stage in June and 40 g m^–2 at ear formation stage in September. The seasonal variation of daily totals of carbon dioxide flux shows that carbon dioxide of about 28 t ha^–1 is fixed by the paddy crop from transplanting to harvesting. Taking into account the water use efficiency, the paddy crop requires water in amounts at least 100 times that of carbon dioxide fixed by photosynthesis. It is noted that the correlation coefficients between carbon dioxide, water vapor and vertical wind velocity have constant values under near neutral and free convective regimes.     

Regulation of glutamine metabolism during the development of Bombyx mori larvae

Waste ammonia is re-assimilated into amino acids via the amide group of glutamine and the amino group of glutamate (i.e. through glutamine synthetase/glutamate synthase pathway) for silk synthesis in the silkworm, Bombyx mori, in the last larval stadium. Glutamine concentration in hemolymph gradually decreased with the progress of the fifth instar and it remained at very low levels during the spinning stage, then followed by a sharp increase at the larval-pupal ecdysis. The changes in glutamine synthetase (GS) activity in silkworm tissues were relatively small through the larval development, while the changes in glutamate synthase (GOGAT) activity, especially in the posterior silk glands, were more drastic. In addition, activities of GOGAT in the tissues were much higher than those of the other enzymes involved in glutamine utilization, suggesting that glutamine pool was regulated mainly by the changes in GOGAT activity. Western blot analysis indicated that the changes in GOGAT protein level correlated with the changes in GOGAT activity. Topical application of a juvenile hormone analogue, methoprene, induced an accumulation of glutamine in the hemolymph of the fifth instar larvae. The levels of GOGAT protein and activity in the tissues of the methoprene treated larvae were much lower than those of the control larvae, whereas the methoprene treatment had no effect on the levels of GS activity. In conclusion, GOGAT expression promoted by reduction of juvenile hormone titer is quite important for enhanced utilization of nitrogen for synthesis of silk protein during the last larval instar.     

The influence of plant carbon dioxide and nutrient supply on susceptibility to insect herbivores

The carbon/nutrient ratio of plants has been hypothesized to be a significant regulator of plant susceptibility of leaf-eating insects. As rising atmospheric carbon dioxide stimulates photosynthesis, host plant carbon supply is increased and the accompanying higher levels of carbohydrates, especially starch, apparently dilute the protein content of the leaf. When host plant nitrogen supply is limited, plant responses include increased carbohydrate accumulation, reduced leaf protein content, but also increased carbon-based defensive chemicals. No change, however, has been observed in the concentration of leaf defensive allelochemicals with elevated carbon dioxide during host plant growth. Insect responses to carbon-fertilized leaves include increased consumption with little change in growth, or alternatively, little change in consumption with decreased growth, as well as enhanced leaf digestibility, reduced nitrogen use efficiency, and reduced fecundity. The effects of plant carbon and nutrient supply on herbivores appear to result, at least in part, from independent processes affecting secondary metabolism.