Permeability of milk protein antigens across the intestinal epithelium in vitro

Degradations by proteolytic enzymes and intestinal epithelial permeability represent two major drawbacks to the transfer of food protein antigens to blood. These steps were studied in vitro for the milk protein antigens beta-lactoglobulin (beta-Lg), alpha-Lactalbumin (alpha-La) and beta-casein (beta-cas). Pepsin-trypsin hydrolysis and permeability in isolated rabbit ileum in Ussing chamber were suited by ELISA and radiolabelled-protein measurement. Pepsin-trypsin hydrolysis showed an increasing resistance in the order beta-cas less than alpha-La less than beta-Lg. The rate of absorption of the antigenic proteins by isolated rabbit ileum was in the same order, and the rate of absorption of the whole proteins (degraded and antigenic forms) was significantly higher for beta-Lg than for alpha-La and beta-cas. These results suggest a selective intestinal permeability for milk protein antigens. This selectivity is probably important in the mechanism of food protein sensitization via the oral route.     

Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.

The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.     

Effect of proteolysis inhibitors on the incorporation of labelled amino acids into proteins]

Role of peptide bond breaks in the incorporation of amino acids into proteins in a "protein--amino acid" system is investigated. For this purpose the incorporation of labelled amino acids into trypsin under the inhibition of its autolysis by a specific inhibitor from soybean and epsilon-amino-caproic acid is studied. The trypsin inhibitor from soybean is found to suppress considerably the incorporation of 14C-glycine, 14C-lysine and 14C-methionine into crystal trypsin and not to affect the incorporation of labelled amino acids into chomotrypsin, papain and carboxypeptidase. Epsilon-Aminocaproic acid inhibited 14C-glycine incorporation into crystal trypsin by 40% and did not change its incorporation level into serum albumin. The dependency of amino acid incorporation level into trypsin on the activity of autolysis in the "protein--amino acid" system is demonstrated.     

白额鹱卵壳的扫描电镜观察

本文报道白额鹱卵壳的壳膜、孔锥层、海绵层、表层等的超微结构,并对卵壳元素进行ＴＮ－５５００能谱分析。     

Nonenzymatic incorporation of prostaglandin A1 into phospholipids in rat liver microsomes

The incorporation of [5,6(n)-3H]prostaglandin A1 (PGA1) and [1-14C]oleic acid into membrane phospholipids of rat liver microsomes was studied. It was shown that PGA1 is incorporated into phospholipids in a much lesser degree than oleic acid. PGA1 is incorporated into phosphatidylethanolamine and, in a lesser degree, into phosphatidylcholine and phosphatidylinositol + phosphatidylserine. The exogenous cofactors of fatty acid acylation (ATP, CoA, Mg2+) exert no marked influence on the incorporation of PGA1 into the phospholipids. PGA1 interacts with isolated rat liver phospholipids; the PGA1-phospholipid conjugate formed is not destroyed in the course of one- or two-dimensional thin-layer chromatography. On the other hand, PGA1 binding to unsaturated phosphatidylcholines is strictly dependent on the phospholipid oxidation index. It is concluded that PGA1 incorporation into rat liver phospholipids is a result of interaction of PGA1 with peroxidized phospholipids.     

Effect of light quality and larval diet on morph induction in the polymorphic caterpillar Nemoria arizonaria (Lepidoptera: Geometridae)

The emerald moth Nemoria arizonaria (Geometridae) is bivoltine, with distinct broods of caterpillars hatching in the spring and summer. Caterpillars of the spring brood develop into mimics of oak catkins, while caterpillars of the summer brood develop into mimics of oak twigs. Previous rearing experiments showed that all caterpillars reared on oak catkins developed into catkin morphs, while all caterpillars reared on oak leaves developed into twig morphs, regardless of temperature or photoperiod. However, those previous rearing experiments did not control the colour of light perceived by the caterpillars independently of their dict. Since wavelengths of light perceived by some species of polymorphic caterpillars can influence their colour, it is possible that morph induction in Nemoria arizonaria is due to the characteristics of light reflected from yellow catkins or green leaves, rather than larval diet itself. The experiments reported here independently varied larval diet and light characteristics to determine if light quality is involved in morph induction. Only larval diet influenced morph induction, since all caterpillars reared on catkins developed into the catkin morph, and all caterpillars reared on oak leaves developed into the twig morph, regardless of whether they perceived yellow light, green light, or were raised in the dark.     

Modeling the hazard of transition into the absorbing state in the illness-death model

The illness-death model is the simplest multistate model where the transition from the initial state 0 to the absorbing state 2 may involve an intermediate state 1 (e.g., disease relapse). The impact of the transition into state 1 on the subsequent transition hazard to state 2 enables insight to be gained into the disease evolution. The standard approach of analysis is modeling the transition hazards from 0 to 2 and from 1 to 2, including time to illness as a time-varying covariate and measuring time from origin even after transition into state 1. The hazard from 1 to 2 can be also modeled separately using only patients in state 1, measuring time from illness and including time to illness as a fixed covariate. A recently proposed approach is a model where time after the transition into state 1 is measured in both scales and time to illness is included as a time-varying covariate. Another possibility is a model where time after transition into state 1 is measured only from illness and time to illness is included as a fixed covariate. Through theoretical reasoning and simulation protocols, we discuss the use of these models and we develop a practical strategy aiming to (a) validate the properties of the illness-death process, (b) estimate the impact of time to illness on the hazard from state 1 to 2, and (c) quantify the impact that the transition into state 1 has on the hazard of the absorbing state. The strategy is also applied to a literature dataset on diabetes.     

The effectiveness of antibiotics on bacteria in biofilms

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.     

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.     

Method of culturing amniotic fluid cells]

The influence of pregnancy term and the concentration of cell suspension, introduced into culture, on the growth of amniotic fluid cells have been studied on 29 specimens. Optimum term of amniotic fluid cells sampling for the best growth was 17 weeks of pregnancy. It is recommended to take into account the concentration of cell suspension in the amniotic fluid before introducing the cells into the culture depending on pregnancy terms.     

Utilization of a biogeochemical model in dendroecology. Application to the Cembro pine

Dendroecology, which is based on annual growth variable measurements, is in possession of data particularly well adapted to analyse the impact of global change on vegetation production. But the classical statistical methods of quantification of tree ring-climate relationship cannot take into account the effect of CO2 increase. Therefore, a biogeochemistry model (BIOME3) has been adapted to these data and then validated on Pinus cembra. The results indicate that the production is reduced by 14% if only the climatic changes are taken into account. If both climatic changes and CO2 increase are taken into account the production is increased by 62%. The direct fertilisation effect of CO2 increase will have more influence on the productivity than the indirect climatic effect.     

Issues related to dust aerosols in the magnesite industry. I. Chamber exposure.

The present paper is an overview of the experimental research into the effects of flue magnesite dust in the magnesite industry in which the raw material (magnesite) is processed into refractory magnesite clinker. The issues related to dust are divided into two problem areas: a) dust aerosol arising in the process of ore mining and consisting largely of magnesite (MgCO3) and b) dust aerosol originating during ore baking in rotatory furnaces and made up mostly of MgO. Thus, larger groups of people become exposed to these aerosols as a result of solid particles escaping into the atmosphere than in the case of occupational exposure. Experimental research carried out on laboratory animals after chamber exposure provided findings on the deposition, retention and elimination of magnesite dust, on impaired balance between magnesium and calcium leading to damage of biological membranes, on how the immune profile or reproduction and embryogenesis is impacted as well as on the possible interaction with sodium salicylate as a result of an impaired acid base balance. These findings are followed up by evidence produced in the course of biological monitoring (Part II).     

Sustainable design indicators: Roadway project as an example

Environmental sustainability has been more examined in the construction industry in recent years. But it is still difficult for engineering designers to incorporate sustainability into their work without practical methods. The design stage is key in the life cycle to integrating sustainability into construction projects. Assessment of sustainable design performance can be an initiative to pursue sustainability.This paper proposes the ratio of items considered/adopted and man-hour spending to measure project sustainable design performance. The two indicators were tested on six roadway projects to validate their applicability on engineering design.The results show that the ratios of items considered and adopted are from 34% to 87%, meaning the many suggested sustainability items can be incorporated into design. The man-hours spent on sustainability are from 2% to 12%, meaning sustainable design initially takes more time than conventional design.The engineering design itself does not cause environmental impact. But the proposed indicators can examine the effort devoted into sustainability in the design stage. This early step helps predict the future environmental performance of designed products.     

Electrophoretic microheterogeneity and subunit composition of the 13S coupling factors of oxidative and photosynthetic phosphorylation.

Two electrophoretically distinguishable species of the 13S coupling factor of oxidative phosphorylation from Alcaligenes faecalis are detectable by standard polyacrylamide gel electrophoresis in the absence of urea, detergents, or any other protein-denaturing reagents. The slower species (type IA) can be converted into the faster species (type IB) by treatment with ATP, and the fast form converts into the slow form when aged at 4 degrees. The enzyme undergoes these conversions both when it is free in solution and when it is membrane bound. The ATP analog adenylyl imidodiphosphate (AMP-PNP) gives the conversion without being hydrolyzed and without causing any apparent change in the mass of the protein, which suggests that the conversion may be a ligand-induced conformational change. Types IA and IB can convert into three other electrophoretically distinguishable species (types IIA, IIB, and III) if the purification procedure involves chromatography on a DEAE-Sephadex column equilibrated in phosphate buffer. These conversions can be prevented if the column is eluted in morpholinoethanesulfonic acid (Mes) buffer and KCl. Type IIA is convertible into type IIB by ATP treatment. Types IA and IB will also convert into types IIA and IIB and finally into type III when aged for extended periods of time at 4 degrees, without a detectable change in mass. Coupling factor activity is lost when type I enzyme converts into type II enzyme, as is the ability of the enzyme to bind to the membrane. However, ATPase activity does not change significantly. The mitochondrial 13S coupling factor shows up to three electrophoretically distinguishable species. The use of phosphate buffer during DEAE-Sephadex chromatography gives conversion of slower species into faster species. ATP treatment does not give interconversions, and aging at 4 degrees gives only a slow dissociation of the enzyme into subunits. The chloroplast 13S coupling factor also shows up to three electrophoretic species. Incubation with ATP does not give interconversions, but a temperature-dependent conversion of the major species into a faster species occurs upon aging. The subunit composition of the three 13S enzymes is very similar by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the major difference being in the number of classes of small polypeptides.     

Synthesis of Chloroplast and Cytosol Ribosomes in Regenerating Acetabularia Cells

The incorporation of ³H-uridine into 80S cytosol and into 70S chloroplast ribosomes has been studied in Acetabularia in order to find out whether or not the nuclei undergo changes in the capability of rRNA formation during differentiation. To reinforce the differentiation process regenerating Acetabularia cells were chosen. Regeneration was induced by amputation of the apical part of the cell. The incorporation into cytosol ribosomes increased as early as 24 h after amputation. The stimulation reaches a maximum three to four days after amputation and does not depend on light at least for three days. It can be prevented, however, by addition of cycloheximide to the culture medium. The stimulation pattern of the uridine incorporation into chloroplast ribosomes is similar to the cytosol ribosomes with the exception that it is delayed by two days. This process is dependent on light and is not inhibited by cycloheximide. The response to amputation seems to be independent of chloroplast energy production as small and large fragments are stimulated to the same degree.     

Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.     

组织培养法快速繁殖川乌头种苗

本研究探讨了川乌头组织培养过程中的外植体诱导建立无菌繁殖系;外植体生长分化和增殖培养基的筛选:生根及驯化移栽等关键技术环节,从而使川乌头通过组培快繁实现大规模育苗成为可能.     

Process of protein transport by the type III secretion system.

The type III secretion system (TTSS) of gram-negative bacteria is responsible for delivering bacterial proteins, termed effectors, from the bacterial cytosol directly into the interior of host cells. The TTSS is expressed predominantly by pathogenic bacteria and is usually used to introduce deleterious effectors into host cells. While biochemical activities of effectors vary widely, the TTSS apparatus used to deliver these effectors is conserved and shows functional complementarity for secretion and translocation. This review focuses on proteins that constitute the TTSS apparatus and on mechanisms that guide effectors to the TTSS apparatus for transport. The TTSS apparatus includes predicted integral inner membrane proteins that are conserved widely across TTSSs and in the basal body of the bacterial flagellum. It also includes proteins that are specific to the TTSS and contribute to ring-like structures in the inner membrane and includes secretin family members that form ring-like structures in the outer membrane. Most prominently situated on these coaxial, membrane-embedded rings is a needle-like or pilus-like structure that is implicated as a conduit for effector translocation into host cells. A short region of mRNA sequence or protein sequence in effectors acts as a signal sequence, directing proteins for transport through the TTSS. Additionally, a number of effectors require the action of specific TTSS chaperones for efficient and physiologically meaningful translocation into host cells. Numerous models explaining how effectors are transported into host cells have been proposed, but understanding of this process is incomplete and this topic remains an active area of inquiry.     

Glucolysis in Pseudomonas putida: Physiological Role of Alternative Routes from the Analysis of Defective Mutants

A number of mutants in which glucolysis is impaired have been isolated from Pseudomonas putida. The study of their behavior shows that this organism possesses a single glucolytic pathway with physiological significance. The first step of the pathway consists in the oxidation of glucose into gluconate. Two proteins with glucose dehydrogenase activity appear to exist in P. putida but the reasons for this duplicity are not clear. The process continues with the formation of 2-ketogluconate which is in turn converted into gluconate-6-phosphate. This is proved by the fact that mutants unable to form gluconate-6-phosphate from 2-ketogluconate show extremely slow growth on glucose or gluconate (generation times are increased more than 100 times). Other possible routes for the conversion of glucose into gluconate-6-phosphate, the glucose-6-phosphate pathway, or the direct phosphorylation of the gluconate formed by glucose oxidation are only minor shunts in P. putida. The Entner-Doudoroff enzymes, which catalyze the conversion of gluconate-6-phosphate into pyruvate and triosephosphate, appear to be essential to grow on glucose and also on gluconate and 2-ketogluconate. A significative role of the pentose route in the catabolism of these substrates is not apparent from this study. In contrast, P. putida strains showing no activity of the Entner-Doudoroff enzymes grow readily on fructose, although there is evidence that this hexose is at least partially catabolized via gluconate-6-phosphate.     

Economic framework for decision making in biological control

Economic analyses are a valuable input into the decision-making process for biological control programs. The challenge though is how to incorporate qualitative risk assessments of biological control programs, or the risk of nontargeted effects into mathematical economic models. A technique known as threshold cost/benefit analysis is presented and an example on how to apply this method is illustrated using the yellow starthistle biological control program. The results show that incorporating uncertainty into the analysis can have a significant impact on the decision to undertake a biological control program.