Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Induction and Accumulation of Heat Shock-Specific Poly(A) RNAs and Proteins in Soybean Seedlings during Arsenite and Cadmium Treatments

Northern blot hybridization analyzes revealed that poly(A⁺) RNAs homologous to eight heat shock (HS)-specific cDNA clones were induced by arsenite (As) or Cd treatments. The mRNAs accumulated slower, and maximum accumulations were consistently lower than HS-induced levels. Prolonged treatment with low concentrations (50-100 micromolar) of As for 6 hours, or Cd for 12 hours, resulted in decreased accumulations of HS-specific mRNAs. This response resembled the `autoregulation' observed during continuous 40°C HS. However, no autoregulation was evident when soybean seedlings were exposed to high concentrations of As (250 micromolar) or Cd (1 millimolar) for 12 hours. The cDNA probe pCE54 detected a second higher molecular weight poly(A⁺) RNA following As or Cd treatments which accumulated concomitantly with the lower molecular weight HS-specific poly(A⁺) RNA. The patterns of low molecular weight HS polypeptides from in vitro translations induced by HS, As, and Cd, and analyzed by one-dimensional and two-dimensional SDS-PAGE, were similar but temporal differences were apparent. In addition to HS proteins, many control proteins were also detected in both in vitro and in vivo labeling patterns from As and, to a lesser extent, Cd treatments. The chemical agents used in this study apparently induced the accumulation and translation of HS messages in vivo but not in the selective manner as observed during HS treatment.     

Investigation of Digestion and Absorption of a Dietary Protein by Using Its Antigenicity as an Index: Gastrointestinal Digestion of Ovalbumin

An attempt was made to use the disappearance of ovalbumin antigen as an index for the analysis of the process of digestion of ovalbumin in vitro and in vivo. The antigenic determinants of ovalbumin were highly sensitive to conformational changes in the protein. Mild heat-treatment and acid-denaturation abolished spontaneously all the reactivities of antigenic determinants. Experiments on protease digestion in vitro showed that native ovalbumin was hardly digested by pepsin at pH 2.7 or by pancreatin at pH 8.0, while acid- or heat-denatured ovalbumin was more easily digested by these proteases. The results suggested that the denaturation of ovalbumin was a rate-limiting step in its digestion, and the disappearance of ovalbumin antigen was a useful index of the initial process of digestion which preceded the proteolytic hydrolysis.

At 1 hr after the administration of an ovalbumin diet to rats, approximately 20% of the ovalbumin antigen administered was detected in the whole contents of the gastrointestinal tract. In the small intestine, the amount of intact ovalbumin comprised about half of the total protein of the intestinal contents. These results suggested that in the stomach and small intestine, denaturation of ovalbumin was a significant rate-limiting step in its digestion process. But after 2hr, little ovalbumin antigen was detected in the small intestine. This disappearance of the antigen from the small intestine cannot be well explained by the in vitro data.     

Effect of chito-oligosaccharide on the oral absorptions of phenolic acids of Flos Lonicerae extract

Phenolic acids, the main active ingredients in Flos Lonicerae extract possess strong antibacterial, antioxidant and antiviral effects, and their contents was higher largely than that of other ingredients such as flavones, but the absolute bioavailability orally was significantly low, which is significant low influencing clinical efficacies of its oral preparations. In the present study, in vitro Caco-2 cell, in situ single-pass intestinal perfusion and in vivo pharmacokinetics study were performed to investigate the effects of COS on the intestinal absorption of phenolic acids. The pharmacological effects such as antiviral activity improvement by COS were verified by MDCK cell damage inhibition rate after influenza virus propagation. The observations from in vitro Caco-2 cell showed that the absorption of phenolic acids in Flos Lonicerae extract could be improved by COS. Meanwhile, COS at the same low, medium and high concentrations caused a significant, concentration-dependent increase in the P_app-value for phenolic acids compared to the control group (p < 0.05), and was all safe for the Caco-2 cells. The observations from single-pass intestinal perfusion in situ model showed that the intestinal absorption of phenolic acids can be enhanced by COS. Meanwhile, the absorption enhancing effect of phenolic acids might be saturable in different intestine sites. In pharmacokinetics study, COS at dosage of 25 mg/kg improved the bioavailability of phenolic acids in Flos Lonicerae extract to the greatest extent, and was safe for gastrointestine from morphological observation. Besides, treatment with Flos Lonicerae extract with COS at dosage of 25 mg/kg prevented MDCK cell damage upon influenza virus propagation better than that of control. All findings above suggested that COS at dosage of 25 mg/kg might be safe and effective absorption enhancer for improving the bioavailability of phenolic acids and the antiviral activity in vitro in Flos Lonicerae extract.     

Interference between leucine, isoleucine and valine during intestinal absorption

1. The reciprocal interference between l-leucine, l-isoleucine and l-valine during absorption was studied in rats both in vivo and with an everted-sac preparation in vitro. 2. After feeding with the amino acids alone there was a considerable increase in their concentration in the intestinal lumen followed by a rapid disappearance, indicating efficient absorption. Absorption was reflected by a high concentration of the respective amino acids in the portal plasma. Isoleucine and valine inhibited the absorption of leucine, and leucine inhibited the absorption of isoleucine and valine. Inhibition of absorption by the interfering amino acid was generally partly overcome after 30–60min., probably through the absorption of the interfering amino acid. At that time the rise in the concentration of the amino acid in portal plasma began. 3. These results were confirmed by experiments in vitro: isoleucine and valine inhibited the absorption rate of leucine, and leucine that of isoleucine and valine. 4. Active absorption of amino acids was rapid at low concentrations and depressed at higher concentrations.     

Regulatory Structure of the Biosynthetic Pathway for the Aspartate Family of Amino Acids in Lemna paucicostata Hegelm. 6746, with Special Reference to the Role of Aspartokinase

Comprehensive studies were made with Lemna paucicostata Hegelm. 6746 of the effects of combinations of lysine, methionine, and threonine on growth rates, soluble amino acid contents, aspartokinase activities, and fluxes of 4-carbon moieties from aspartate through the aspartokinase step into the amino acids of the aspartate family. These studies show that flux in vitro through the aspartokinase step is insensitive to inhibition by lysine or threonine, and confirm previous in vitro data in establishing that aspartokinase in vivo is present in two orders of magnitude excess of its requirements. No evidence of channeling of the products of the lysine- and threonine-sensitive aspartokinases was obtained, either form of the enzyme alone being more than adequate for the combined in vivo flux through the aspartokinase step. The marked insensitivity of flux through the aspartokinase step to inhibition by lysine or threonine strongly suggests that inhibition of aspartokinase by these amino acids is not normally a major factor in regulation of entry of 4-carbon units into the aspartate family of amino acids. Direct measurement of fluxes of 4-carbon units demonstrated that: (a) Lysine strongly feedback regulates its own synthesis, probably at the step catalyzed by dihydrodipicolinate synthase. (b) Threonine alone does not regulate its own synthesis in vivo, thereby confirming previous studies of the metabolism of [¹⁴C]threonine and [¹⁴C]homoserine in Lemna. This finding excludes not only aspartokinases as an important regulatory determinant of threonine synthesis, but also two other enzymes (homoserine dehydrogenase and threonine synthase) suggested to fulfill this role. Complete inhibition of threonine synthesis was observed only in the combined presence of accumulated threonine and lysine. The physiological significance of this single example of apparent regulation of flux at the aspartokinase step, albeit under unusually stringent conditions of aspartokinase inhibition, remains to be determined. (c) Isoleucine strongly inhibits its own synthesis, probably at threonine dehydratase, without causing compensatory reduction in threonine synthesis. A fundamentally changed scheme for regulation of synthesis of the aspartate family of amino acids is presented that has important implications for improvement of the nutritional contents of these amino acids in plants.     

Effects of Meat Cooking,and of Ingested Amount,on Protein Digestion Speed and Entry of Residual Proteins into the Colon: A Study in Minipigs

The speed of protein digestion impacts on postprandial protein anabolism. After exercise or in the elderly, fast proteins stimulate protein synthesis more efficiently than slow proteins. It has been shown that meat might be a source of fast proteins. However, cooking temperature, acting on the macrostructure and microstructure of the meat could affect both the speed, and efficiency, of protein digestion. This study aims to evaluate, in vivo, the effect of meat cooking on digestion parameters, in the context of a complete meal. Six minipigs fitted with an ileal cannula and an arterial catheter were used. In order to measure the true ileal digestibility, tested meat was obtained from a calf, the muscle proteins of which were intrinsically labelled with ¹⁵N-amino acids. Three cooking temperatures (60, 75 and 95°C; core temperature for 30 min), and three levels of intake (1, 1.45, and 1.90 g protein/kg body weight) were tested. Following meat ingestion, ileal digesta and arterial blood were collected over a 9-h period. The speed of digestion, evaluated from the kinetics of amino acid appearance in blood within the first 3 h, was greater for the cooking temperature of 75°C, than for 60 or 95°C. The true ileal digestibility, which averaged 95%, was not affected by cooking temperature or by the level of meat intake. The amino acid composition of the digesta flowing at the ileum was not affected by cooking temperature. These results show that cooking temperature can modulate the speed of meat protein digestion, without affecting the efficiency of the small intestinal digestion, and consequently the entry of meat protein residues into the colon.     

In vivo and in vitro effects of cadmium on selected enzymes in different organs of the fish Barbus conchonius ham. (Rosy Barb)

1. Enzyme modulation by cadmium in selected organs of the fish, Barbus conchonius (rosy barb), was investigated in vivo (48 hr exposure to 12.6 mg/1 cadmium chloride) and in vitro (10⁻⁶M cadmium chloride).2. The acetylcholinesterase (AchE) activity was depressed in the gills but stimulated in the skeletal muscles and brain in vivo. The hepatic, branchial, and renal acid phosphatase (AcP) activity decreased marginally in vivo but it was significantly increased in the gut and ovary. In vitro, except for the liver, the AcP activity was depressed in the selected organs. Collaterally, gut alkaline phosphatase (A1P) was significantly inhibited but a pronounced stimulation was noted in the kidneys and ovary in vivo. In vitro, the AIP activity was conspicuously elevated in the kidneys and gut, and moderately in the gills.3. Cadmium inhibited the glutamate-oxaloacetate and glutamate-pyruvate transaminases (GOT and OPT) in the liver, gills and kidneys in vivo. In vitro, the GOT and GPT activities were decreased in the liver, gills and kidneys. The lactic dehydrogenase (LDH) was significantly stimulated by Cd in the heart in vivo but in vitro the metal inhibited the enzyme in the gills.4. Enzymes in the liver, followed by those in the kidneys and gills seem to be most seriously affected by Cd poisoning in this fish.     

Stomach digestion and its effect upon protein hydrolysis in the intestine of rainbow trout (Salmo gairdneri Richardson)

• 1.1. The in vitro digestion of soya protein by pancreatic proteases of the trout was measured following various conditions of stomach digestion.
• 2.2. Peptic hydrolysis results in an increase of small peptides.
• 3.3. Acid treatment and peptic proteolysis do not affect the degradation of insoluble proteins or the formation of free amino acids during intestinal digestion, but they do lead to a significant shift from soluble polypeptides to di- and oligopeptides.

     

Cadmium distribution and chemical fate in soybean plants

The distribution and chemical behavior of Cd²⁺ in tissues and its chemical form in xylem water of soybean plants (cv. Williams) were investigated. Following root absorption, Cd is strongly retained by roots, with only 2% of the accumulated Cd being transported to leaves; as much as 8% was transported to seeds during seed filling. In vivo xylem exudates contained two anionic Cd complexes in addition to inorganic forms of Cd. Once accumulated in root and leaf tissues, Cd rapidly equilibrated between the insoluble, soluble, and organelle fractions. Of the solubles, which contain 50% of the Cd, >50% was associated with components of >10,000 molecular weight, and <8% was associated with <500 molecular weight components. Cadmium accumulated in soybean seeds was primarily associated with cotyledons. Fractionation of seeds showed the soy proteinate and soy whey to contain 32 and 50% of the accumulated Cd, respectively.     

Sites of synthesis of plasma proteins in the foetal rat

1. The foetal rat of 16 or more days incorporates ¹⁴C-labelled amino acids into all the demonstrable plasma protein fractions in vivo. 2. Slices of foetal rat liver incubated in vitro incorporate ¹⁴C-labelled amino acids into the main plasma protein fractions, including the foetal-specific `post-albumin'. 3. Slices of placenta are unable to incorporate ¹⁴C-labelled amino acids into plasma proteins in vitro. 4. Liver slices from maternal rats incubated in vitro incorporate ¹⁴C-labelled amino acids into plasma proteins. The presence of post-albumin cannot be demonstrated after incubation. 5. Liver slices from foetal rats, but not from adult rats, contain demonstrable amounts of haemoglobin into which ¹⁴C-labelled amino acids are incorporated.     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices

Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R² = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.     

Cadmium elevates level of protein, amino acids and alters activity of proteolytic enzymes in germinating rice seeds

When seeds of two rice cvs. Ratna and Jaya were germinated under increasing levels of cadmium nitrate (0, 100 and 500 μM) in the medium, a marked decrease in germination percentage was observed with Cd treatments, as compared to controls. There was more absorbed Cd in embryo axes than in endosperms. More uptake resulted with increasing Cd levels in the growth medium in embryo axes. In both rice cultivars, during a germination period of 0 – 120 h, an increased level of protein as well as free amino acids was noted in Cd treatments. Protease activity in general decreased in both embryo axes as well as endosperms due to Cd treatment. In vitro studies showed an enhancement in protease activity in Cd treatments at low Cd levels (50–100 μM), whereas concentrations above this caused inhibition in enzyme activity. Under 500 μM Cd treatments in vivo there was about 30 to 50 percent decline in leucine aminopeptidase (LAP) activity in endosperms, however, carboxypeptidase activity showed a marked increase in endosperms beyond 24 h under Cd treatments. In embryo axes of germinating seeds there was always a decline in peptidase activities, under the influence of cadmium. The leucine amino peptidase and protease activity were always greater in embryo axes in cv. Ratna than cv. Jaya. However, the carboxypeptidase activity was higher in Jaya when compared to Ratna in endosperms under Cd treatments. The results suggest possible suppression of protease and peptidase activities due to Cd treatments in germinating rice seeds leading to altered levels of protein and amino acids.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Protein digestion in cereal aphids (Sitobion avenae) as a target for plant defence by endogenous proteinase inhibitors

Gut extracts from cereal aphids (Sitobion avenae) showed significant levels of proteolytic activity, which was inhibited by reagents specific for cysteine proteases and chymotrypsin-like proteases. Gut tissue contained cDNAs encoding cathepsin B-like cysteine proteinases, similar to those identified in the closely related pea aphid (Acyrthosiphon pisum). Analysis of honeydew (liquid excreta) from cereal aphids fed on diet containing ovalbumin showed that digestion of ingested proteins occurred in vivo. Protein could partially substitute for free amino acids in diet, although it could not support complete development. Recombinant wheat proteinase inhibitors (PIs) fed in diet were antimetabolic to cereal aphids, even when normal levels of free amino acids were present. PIs inhibited proteolysis by aphid gut extracts in vitro, and digestion of protein fed to aphids in vivo. Wheat subtilisin/chymotrypsin inhibitor, which was found to inhibit serine and cysteine proteinases, was more effective in both inhibitory and antimetabolic activity than wheat cystatin, which inhibited cysteine proteases only. Digestion of ingested protein is unlikely to contribute significantly to nutritional requirements when aphids are feeding on phloem, and the antimetabolic activity of dietary proteinase inhibitors is suggested to result from effects on proteinases involved in degradation of endogenous proteins.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

Puromycin aminonucleoside increases the synthesis of ribonuclease inhibitor and other proteins in kidney

The incorporation of labelled amino acids into proteins was measured in vivo in the kidney of control rats and rats that received puromycin aminonucleoside. There was an increase in the synthesis of kidney proteins after the aminonucleoside that was similar to the increased synthesis previously observed in cell-free and slice preparations. The increased synthesis in vivo and in vitro especially involved proteins of the prealbumin fraction of average molecular weights of approx. 50 000, 35 000, 25000, 18 000, and 10 000. The largest of these proteins was identified as kidney ribonuclease inhibitor and additional evidence was obtained for the increased synthesis of the kidney inhibitor after aminonucleoside.     

Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas)

Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gigas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.