Iron removal from transferrin by a cell-free amphibian system.

1. Transferrin-bound iron was released by a cell-free solution prepared from Rana catesbeiana immature erythrocytes, showing that cellular integrity is not necessarily a requirement for this process as previously thought. 2. The biological molecule(s) involved in the removal of iron from transferrin is relatively small.     

Competitive binding of iron by transferrins from different vertebrates.

1. A competitive dialysis technique has been used to study the relative affinities of the two iron-binding sites on transferrin molecules and the relative binding strengths of transferrins isolated from plasma of different species. 2. The comparisons were extended to include desialylated human transferrin, ovotransferrin, and a cyanogen bromide fragment of the latter. 3. Although the results of bilateral experiments could generally be accounted for in terms of the theory of independent sites, there were some exceptions, and cyclic comparisons were inconsistent. 4. All the comparisons made were compatible with a model in which site-interaction occurred, but it was not possible to decide whether the sites were intrinsically identical or not. For most species this corresponded to positive cooperativity, but for rabbit it was negative. 5. The average affinity of transferrin for iron depended on species, but the variation was never more than about one order of magnitude. 6. No effect on the binding constants for human transferrin could be detected when the sialic acid residues were removed. 7. The fragment of ovotransferrin competed fairly effectively with the native molecule for iron, although the average relative affinity was only about 1:15. 8. The relative binding of iron by ovotranferrin and human transferrin was affected little when bicarbonate anion was replaced by oxalate, although the ratio of the two binding constants for ovotranferrin increased.     

Lipid biosynthesis in amphibian oocyte and embryonic cell-free preparations

• 1.1. The utilization of [2-³H]glycerol-3-phosphate in the synthesis of lipids during early embryogenesis was studied in cell-free preparations from oocytes or embryos of Bufo arenarum Hensel
• 2.2. The precursor was incorporated in all stages of development up to gill circulation, which indicates that oocytes and embryos have the enzymatic machinery necessary to synthesize at least part of their own lipids.
• 3.3. A significant decrease in the labeling of most lipids took place after fertilization, especially in gastrulas, but at gill circulation lipid synthesis was highly stimulated.
• 4.4. The incorporation pattern is similar in unfertilized oocyte, fertilized oocyte and gastrulas, where phosphatidylglycerol has the highest amount of radioactivity. At gill circulation stage phosphatidylethanolamine and neutral lipid biosynthesis also became significant.
• 5.5. The results suggest a different regulation of the biosynthetic lipid routes through the appearance of new enzymes or modulators of preexisting enzymes during amphibian development.

     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Ribonucleotide reductase activity during amphibian development.

Uniformly labeled [3H] uridine is incorporated into DNA by dissociated Pleurodeles blastulae; the label is found in cytosine and to a much lesser extent in thymine. Ribonucleotide reductase activity cannot be detected in full grown oocytes of Xenopus and Pleurodeles, but is present in unfertilized egg. The enzyme is synthesized (or activated) when maturation is induced in Xenopus oocytes by in vitro hormonal treatment. The enzymatic activity increases after fertilization and reaches a peak at the 2--4 cell stage; it decreases at the blastula, gastrula and neurula stages to the low level initially present in unfertilized eggs. The enzyme is no longer detectable in swimming tadpoles. Addition of hydroxyurea (1 mg/ml) to fertilized eggs leads to complete loss of ribonucleotide reductase activity: cycloheximide (20 mug/ml) inhibits the rise in activity characteristic of early cleavage, while actinomycin D (20 mug/ml) has no effect. The significance of these results in discussed.     

Comparison of ACE activity in amphibian tissues: Rana esculenta and Xenopus laevis

Angiotensin converting enzyme (ACE) is the dipeptidyl-carboxypeptidase of the renin-angiotensin system involved in the control of blood pressure and hydromineral metabolism. It converts angiotensin I to angiotensin II, the biologically active octapeptide. Angiotensin converting enzyme-like activity has been demonstrated in a wide range of vertebrates. The presence of ACE was investigated in tissues of two amphibian species, the frog Rana esculenta and the toad Xenopus laevis. ACE activities were determined by specific substrate hydrolysis in gut, gonads, lung, kidney, heart, liver, skin, erythrocytes, and muscle homogenates and plasma by means of high performance liquid chromatography. Significant ACE activity was found in gut, gonads, lung and kidney, while that in heart, liver, skin, erythrocytes, muscle, and plasma was very low. Testis of toad contained the highest ACE activity, while that in erythrocytes of male and female frogs was notable.     

Differences between human and rabbit transferrins.

Rabbit reticulocyte incorporation of iron from rabbit transferrin was independent of transferrin iron saturation but uptake from human transferrin was saturation dependent. Unlike human transferrin, rabbit transferrin does not surrender its iron from any unique preferred iron-binding site and can be described as functionally homogeneic. The two proteins also differ in their acid-base iron-binding properties. One human transferrin iron binding site retains an ability to bind iron at somewhat acid pH but this property is not shared by rabbit transferrin.     

Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems

The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and full soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 microM. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the Km of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.     

Comparison of O2(-)-producing activity of guinea-pig eosinophils and neutrophils in a cell-free system.

1. The NADPH-dependent superoxide (O2-) production in a cell-free system of guinea-pig eosinophils was studied, comparing the eosinophils with neutrophils. 2. Eosinophils produced 2.2-fold more O2- than neutrophils in sonicated and intact cells. 3. The subcellular fractionation experiments showed that the O2- production was dependent on the cooperation between the membrane and cytosol fractions. 4. The cross-mixing experiments indicated that the NADPH oxidase-activating activity of the eosinophil cytosol was about 2-fold greater than that of the neutrophil cytosol. 5. These results suggest that the difference in the O2(-)-producing activity between eosinophils and neutrophils is associated with the difference in cytosolic factors necessary for the activation of NADPH oxidase.     

Characterization of Xanthomonas campestris pv. pruni strains from different hosts by pathogenicity tests and analysis of whole-cell fatty acids and whole-cell proteins

Strains of Xanthomonas campestris pv. pruni obtained from Prunus armeniaca. P. domestica, P. persica and P. salicina in different geographical areas were compared for pathogenicity, fatty acid and wholecell protein analysis. Four strains, one per each host plant, were inoculated at the same time, on the foliage of P. armeniaca, P. avium, P. persica and P. salicina cultivars . Mean content of fatty acids of X.c. pv. pruni strains were also compared with those of many strains of X.c. pv. campestris , pv. graminis , pv. hyacinthii , pv. pelargonii and pv. vasculorum. Strains showed a remarkable homogeneity in fatty acids content and whole-cell protein profiles and principal component and cluster analysis did not reveal any grouping according to original host or geographical origin. However, X.c . pv. pruni strains can be grouped apart from the other X. campestris pathovars. There appears to be no pathogenic specialization among the strains tested, however, they varied in aggressiveness to host plants and host plant in susceptibility. The most of the strains were able to cross-infect species other that from where they were originally isolated, although, P. avium did not show any symptom of disease. P. persica cv. Sentry and P. salicina cv. Globe Sun, recently licensed as resistant to X.c. pv. pruni. were infected, although to a lesser extent, by some strains.     

Cytotoxic activity of lymphocytes. VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes.

Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M_r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N-ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.     

Comparison of different carbon ink based screen-printed electrodes towards amperometric immunosensing

The aim of the present work was to make amperometric immunosensors based on the principle of enzyme-linked immunosorbent assay (ELISA). For this purpose, screen-printed electrodes (SPEs) were fabricated using various carbon inks (commercially available inks Gwent, Acheson, Eltecks and two homemade inks PSG & PVCG) to determine the best ink in realizing immunosensors. Amperometric immunosensors made by different carbon inks were compared with standard ELISA in terms of total assay time, amount of biological materials used and sensitivity of detection. A model system containing rabbit anti-mouse immunoglobulin G (RαMIgG) as the capturing antibody, mouse IgG (MIgG) as antigen and alkaline phosphatase conjugated RαMIgG as revealing antibody was used. In these studies, 1-naphthyl phosphate was used as substrate. The experiments done include electrochemical characterization of electrodes, optimization of dilutions of antibodies, immobilization of antibody on the electrode were carried out. The minimum detection limit for the best results of MIgG determination were obtained on screen-printed electrode made by Gwent carbon ink and PSG carbon ink, with a detection limit of 1.0 and 2.0 ng/ml respectively. The time required for detection of mouse IgG was 40 min for SPEs. By using the conventional spectrophotometric method (ELISA method), the minimum detection limit for the MIgG (antigen) detection was 50 ng/ml and the time required for analysis was found to be 140 min.     

Protective activity of a cell-free Klebsiella vaccine in relation to different Klebsiella pneumoniae serovars

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial cells with hydroxylamine, for protecting mice from Klebsiella septic infection caused by the homologous serovar and 9 heterologous serovars of K. pneumoniae was studied. The newly developed preparation was found capable of stimulating immunity not only to the homologous K. pneumoniae serovar, but also to other K. pneumoniae heterologous serovars: K1, K9, K11, K16, K20, K61. The protective capacity of the preparation with respect to these serovars was not inferior to that of the vaccines prepared by the same method from the corresponding homologous strains. The capacity of the vaccine to protect mice from Klebsiella sepsis was manifested irrespective of the virulence of the strains used for challenge.     

Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity.

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.