Possible involvement of an enzymatic system for superoxide generation in lepidopteran larval haemolymph

The superoxide generative system in Pseudaletia separata larval haemolymph plasma was shown to consist of at least two components, low (LMF) and high (HMF) molecular weight factors using ultrafiltration, dialysis, and gel filtration. The LMF was concluded to be a molecule(s) smaller than 5 kDa while the HMF to be a protein(s) larger than 100 kDa. The total amount of superoxide produced depended on the amount of LMF and was independent of that of HMF added to the reaction mixture. Both the LMF and HMF were required for O₂^? generation. From results in the present article, it was hypothesized that the LMF was a substrate(s) discharging electrons and HMF was an enzyme(s) to mediate the electron transfer to O₂ forming O₂^?. Superoxide production was detected in the haemolymph plasma of 5 lepidopteran species in addition to P. separata. It was concluded that superoxide production is a common phenomenon at least in these lepidopterans. © 1995 Wiley-Liss, Inc.     

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.     

Studies of in vitro γ-interferon production in coeliac disease as a response to gliadin peptides

The effects of certain fractions of a peptic-tryptic-pancreatinic (PTP) digest of wheat gliadin of synthetic peptides on the production of gamma interferon (γ-IFN) in cultures of whole blood from adult patients with coeliac disease (CD) have been studied using a sandwich enzyme immunoassay. The most active peptides were fraction 9, its two principal sub-fractions (sub-fractions 1 and 2) and a synthetic peptide of sequence RPQQPYPQPQPQ (peptide V) corresponding to the principal peptide obtained from reversed-phase HPLC of fraction 9. Results with blood from the control group of subjects also indicated some response to these antigens, in most cases at similar levels to those observed with the coeliacs. Fraction 1 of the PTP digest and the other nine synthetic peptides tested were inactive with both coeliacs and controls. These results are in agreement with the results of in vivo and in vitro toxicity tests. They provide evidence of a link between toxicity and cell-mediated immune response in CD, and suggest that peptide V represents one of the active parts of the gliadin molecule.     

Microencapsulation of linoleic acid with low- and high-molecular-weight components of soluble soybean polysaccharide and its oxidation process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37 degrees C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.     

Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods

Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with beta-(1-->4)-D-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645+/-11)x 10(3)g/mol and a radius of gyration, Rg, of (23.5+/-2.8)nm. By comparing R(g) with the hydrodynamic radius, Rh (21.1+/-0.5 nm) obtained from DLS, the structural parameter rho (Rg/Rh) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287+/-18)x 10(3)g/mol but a similar radius of gyration (23.2+/-1.7 nm). The structure parameter rho of GPase/SSPS was higher (rho=1.3) because of a smaller hydrodynamic radius (17.7+/-1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation (rho=1.7-2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains.     

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.     

Purification and biochemical characterization of human lymphocyte mitogenic factor (LMF).

Supernatants of human T lymphocytes stimulated by TT antigen release two factors that induce mitogenesis in autologous and allogeneic B lymphocytes. These factors are precipitated by 60% ammonium sulfate and 50% ethanol, and are both destroyed by heating to 70 degrees C for 5 min. By equilibrium ultracentrifugation there was a peak of mitogenic activity in the fraction with a specific gravity of 1.3147 corresponding to a partial specific volume of 0.761. After ultrafiltration through an Amicon XM50 membrane, the concentrate was chromatographed on a Sephadex G-200 column. Mitogenic activity was found only in the post-albumin fraction. When the post-albumin fraction was run on an isoelectrofocusing column, two distinct mitogenic factors were identified. The major peak of mitogenic activity (LMF) had a pI of 6.68 +/- 0.05 and the minor peak (MMF) had a pI OF 7.27 +/- 0.05. Amino acid analysis of LMF identified it as a protein and PAGE showed that LMF probably was a tetramer with a m.w. of 80,000.     

Stimulation of nonspecific resistance in mice with high- and low-molecular fractions of endotoxin on 2 models of Shigella infection]

The capacity of S. paratyphi B endotoxin and its high molecular (HMF) and low molecular (LMF) fractions to stimulate the resistance of mice to shigella infection in intraperitoneal and intranasal challenge was investigated. The HMF developed high activity after the intraperitoneal injection (efficiency index--EI = 2.8). All the preparations were less active on the lung model (EI = 1.6--1.7). Materials of this work lead to the conclusion that in studying the phenomenon of nonspecific resistance induction conditions providing antitoxic and antiinfectious resistance should be considered separately.     

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion

Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.     

Seedling growth strategies in Bauhinia species: comparing lianas and trees

BACKGROUND AND AIMS: Lianas are expected to differ from trees in their growth strategies. As a result these two groups of woody species will have different spatial distributions: lianas are more common in high light environments. This study determines the differences in growth patterns, biomass allocation and leaf traits in five closely related liana and tree species of the genus Bauhinia. METHODS: Seedlings of two light-demanding lianas (Bauhinia tenuiflora and B. claviflora), one shade-tolerant liana (B. aurea), and two light-demanding trees (B. purpurea and B. monandra) were grown in a shadehouse at 25% of full sunlight. A range of physiological, morphological and biomass parameters at the leaf and whole plant level were compared among these five species. KEY RESULTS: The two light-demanding liana species had higher relative growth rate (RGR), allocated more biomass to leaf production [higher leaf mass fraction (LMF) and higher leaf area ratio (LAR)] and stem mass fraction (SMF), and less biomass to the roots [root mass fraction (RMF)] than the two tree species. The shade-tolerant liana had the lowest RGR of all five species, and had a higher RMF, lower SMF and similar LMF than the two light-demanding liana species. The two light-demanding lianas had lower photosynthetic rates per unit area (A(area)) and similar photosynthetic rates per unit mass (A(mass)) than the trees. Across species, RGR was positively related to SLA, but not to LAR and A(area). CONCLUSIONS: It is concluded that the faster growth of light-demanding lianas compared with light-demanding trees is based on morphological parameters (SLA, LMF and LAR), and cannot be attributed to higher photosynthetic rates at the leaf level. The shade-tolerant liana exhibited a slow-growth strategy, compared with the light-demanding species.     

Comparison of antitumor activities of different polysaccharide fractions from the stems of Dendrobium nobile Lindl

The antitumor activities of extracted polysaccharide fractions from the stems of Dendrobium nobile Lindl were investigated. Polysaccharides were sequentially extracted from the stems of D. nobile to obtain three fractions, i.e. water extract fraction (DNP-W), 5% NaOH extract fraction (DNP-OH) and 5% HCl extract fraction (DNP-H). Further the DNP-W was isolated to give six sub-fractions (DNP-W1, DNP-W2, DNP-W3, DNP-W4, DNP-W5 and DNP-W6) by anion-exchange chromatography. The monosaccharide profile, protein content, uronic acid content, total carbohydrate content, viscosity and molecular weight of nine polysaccharide fractions were analyzed. Both the in vivo and in vitro antitumor activities of nine polysaccharide fractions were evaluated and compared. Results indicated that DNP-W1 and DNP-W3 exhibited high antitumor activities against Sarcoma 180 in vivo and HL-60 in vitro. The results suggested that DNP-W1 and DNP-W3 could be considered as an effective natural antitumor source.     

Conversion of biomass into 5-hydroxymethylfurfural using solid acid catalyst

5-Hydroxymethylfurfural (HMF) was produced from monosaccharide (fructose and glucose), polysaccharide (inulin) and the Jerusalem artichoke juice by a simple one-pot reaction including hydrolysis and dehydration using solid acid under mild condition. Hydrated niobium pentoxide (Nb(2)O(5)·nH(2)O(2)) after pretreatment showed high catalytic activities for dehydration of mono- and polysaccharide to HMF at 433 K in water-2-butanol (2:3 v/v) biphasic system, giving high HMF yield of 89% and 54% from fructose and inulin, respectively. The HMF yield was up to 74% and 65% when inulin and Jerusalem artichoke juice were hydrolyzed by exoinulinase. The solid acid made the process environment-friendly and energy-efficient to convert carbohydrates into bio-fuels and platform chemicals.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Functional analysis of mononuclear cells infiltrating into tumors. V. A. soluble factor involved in the regulation of cytotoxic/suppressor T cell infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of cytotoxic/suppressor T lymphocytes in tumor tissues. We found that tumor-infiltrating helper/inducer T cells isolated from T-9 gliosarcoma-sensitized rats between 4 and 6 days after T-9 gliosarcoma inoculation produced a lymphocyte migration factor (LMF) during in vitro culture. Four peaks of LMF activity (A through D) were detected upon fractionation of LMF by using a Mono Q anion exchange column chromatography. Peak C exhibited the strongest activity among the four peaks of LMF. The action of peak C was chemotactic, but not chemokinetic. Peak C had an isoelectric point of 8.0 and a Mr of 26,000 Da. Only cytotoxic/suppressor T cells were found to be sensitive to peak C in vitro as well as in vivo. It is thus likely that peak C is responsible for the infiltration of cytotoxic/suppressor T cells into tumor tissues. The infiltration of lymphocytes into tumor tissues might also be regulated by the expression of lymphocyte sensitivity for LMF. The target molecule for LMF at 4 days may involve an asparagine-linked oligosaccharide.     

Synthesis and in vitro cytotoxicity of cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV)-peptide bioconjugates

The synthesis and characterization of four Pt(IV)-peptide conjugates, containing one or two peptides in the axial position, designed for the purpose of targeted drug delivery to tumor cells, are described. The precursor cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV) was coupled in the last step of standard solid-phase peptide synthesis (SSPS) with an analogue of neurotensin (pseudo-neurotensin = Lys-Lys-Pro-Tyr-Ile-Leu) and with octreotate (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH), an analogue of somatostatin, respectively. For all peptides, the SSPS reactions afforded both mono- and diconjugated Pt-peptide species, which were separated and purified by RP-HPLC. The two couples of conjugates, together with the precursor, were tested as cytotoxic agents towards different cancer cell lines. In general all conjugates are good inhibitors of cellular proliferation when compared to a nontargeting platinum(IV) parent compound, so that its relatively low cytotoxicity is greatly improved by addition of the peptides.     

Quantitative trait loci affecting growth-related traits in wild barley (Hordeum spontaneum) grown under different levels of nutrient supply

The genetic basis of phenotypic plasticity of relative growth rate (RGR), its components and associated morphological traits was studied in relation to nutrient limitation. In all, 140 F(3) lines from a cross, made between two Hordeum spontaneum (wild barley) accessions sampled in Israel, were subjected to growth analysis under two nutrient levels. Quantitative trait loci (QTLs) were detected for RGR and three of its components, leaf area ratio (LAR), specific leaf area and leaf mass fraction (LMF). Indications for close linkage (potential pleiotropy) were found, for example, for LAR and LMF. An interesting case is on chromosome 6, at which QTLs for RGR and seed mass were detected in the same region. These QTLs had opposite additive effects, supporting earlier results that plants growing from lighter seeds had a higher RGR. Only two QTLs were significant under both nutrient conditions, suggesting large QTL x environment interactions for most traits. For 21 out of 26 QTLs, however, the additive genetic effect was of identical sign in both nutrient environments, but reached the significance threshold in only one of them. Nevertheless, some QTLs detected in one of the two environments had virtually no effect in the other, and QTLs for plasticity were detected for RGR, LAR and LMF, as well as for some morphological traits. QTLs with opposite effects under high and low nutrients were not found. Thus, at the genetic level, there was no evidence for a trade-off between faster growth at high versus low nutrient levels.     

印度块菌提取物抗氧化活性的研究

对印度块菌Tuber indicum子实体的提取物,包括55%乙醇粗提物(ECE)、石油醚提取物(PEF)和乙酸乙酯提取物(EAF)的清除DPPH自由基和羟基自由基能力、铁离子鳌合能力以及各提取物的总酚含量等进行了研究和测定,结果显示,3种提取物的清除自由基能力和铁离子鳌合能力具有显著差异(P0.05);ECE对DPPH自由基的清除活性最高,其EC50值为1.61g/L;EAF对羟基自由基及铁离子表现出较强的清除或螯合的能力,其EC50值分别为3.31g/L和0.70g/L;EAF的总酚含量(2.964mg GAE/g提取物)最高,其次是ECE,总酚含量为(2.618mg GAE/g提取物);PEF的清除自由基和铁离子鳌合能力较差,其总酚含量也最低(1.124mg GAE/g提取物);总酚含量与印度块菌提取物清除自由基以及鳌合铁离子的能力密切相关。     

Isolation and characterization of copper-binding sites of human ceruloplasmin

A tryptic cleavage procedure was used to obtain stable copper-containing peptide regions of human ceruloplasmin. Tryptic digestion-derived copper peptides were fractionated by gel filtration chromatography, yielding two fractions, one with an apparent molecular weight of 11000 and the other 1000. The high molecular weight fraction (11K fraction) was found to contain 50% of the copper atoms of the ceruloplasmin molecule and behaved as a single copper-containing component by gel filtration chromatography and by isoelectric focusing. The low molecular weight fraction (1K fraction) was found to be a mixture of three or four copper peptides by isoelectric focusing. Acrylamide gel electrophoresis studies, amino acid composition analysis and terminal amino acid determinations showed the 11K fraction to be a complex composed of at least three peptides arising from different regions of the ceruloplasmin molecule. Two of the peptides of the 11K complex appear to be derived from the 19K fragment of the ceruloplasmin molecule (18); one peptide in the complex appears to correspond to the aspartic acid-rich portion, residues 7-30, and the other to the histidine-rich portion, residues 103-157. Most preparations of ceruloplasmin are reported to consist of three non-covalently linked fragments that have molecular weights of 67K, 50K and 19K. Dwulet and Putnam (20) proposed a model for the sequence structure of ceruloplasmin where the molecule exhibits a three-fold repeat pattern of two alternating structures, here termed X and Y.(ABSTRACT TRUNCATED AT 250 WORDS)     

INDUCTION OF ASTER FORMATION AND CLEAVAGE IN EGGS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS BY INJECTION OF SPERM COMPONENTS*

Studies were made on which components of sperm were able to induce aster formation and cleavage of eggs of the sea urchin Hemicentrotus pulcherrimus. The sperm components were separated by homogenization and centrifugation into the following 3 fractions: the head-midpiece, midpiece and tail. The head-midpiece fraction was then divided into 2 sub-fractions, the centriole sub-fraction and the centriole-free sub-fractions. Each fraction was injected into unfertilized eggs and after 15–30 min the eggs were inseminated. The ability of a fraction or a sub-fraction to induce aster formation and cleavage was deduced from the frequency of multipolar cleavage. The head-midpiece fraction and the centriole sub-fraction were effective in inducing aster formation and cleavage, but the other fractions were not. It was concluded that isolated centrioles from sea urchin sperm act as division centers in the egg.     

Fractionation of distillers dried grains with solubles (DDGS) by sieving and winnowing

Four commercial samples of distillers dried grains with solubles (DDGS) were sieved. All sieved fractions except for the pan fraction, constituting about 90% of original mass, were then winnowed with an air blast seed cleaner. Sieving was effective in producing fractions with varying composition. As the particle size decreased, protein and ash contents increased, and total carbohydrate (CHO) decreased. Winnowing sieved fractions was also effective in shifting composition, particularly for larger particle classes. Heavy sub-fractions were enriched in protein, oil and ash, while light sub-fractions were enriched for CHO. For protein, the combination of the two procedures resulted in a maximum 56.4% reduction in a fraction and maximum 60.2% increase in another fraction. As airflow velocity increased, light sub-fraction mass increased, while the compositional difference between the heavy and light sub-fractions decreased. Winnowing three times at a lower velocity was as effective as winnowing one time at a medium velocity. Winnowing the whole DDGS was much less effective than winnowing sieved fractions in changing composition, but sieving winnowed fractions was more effective than sieving whole DDGS. The two combination sequences gave comparable overall effects but sieving followed by winnowing is recommended because it requires less time. Regardless of combinational sequence, the second procedure was more effective in shifting composition than the first procedure.