The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Abundantly Present miRNAs in Milk-Derived Extracellular Vesicles Are Conserved Between Mammals.

Mammalian milk is not only a source of nutrition for the newborn, but also contains various components that regulate further development. For instance, milk is an abundant source of microRNAs (miRNAs), which are evolutionary conserved small non-coding RNAs that are involved in post-transcriptional regulation of target mRNA. MiRNAs present in milk can occur in extracellular vesicles (EVs), which are nanosized membrane vesicles released by many cell types as a means of intercellular communication. The membrane of EVs protects enclosed miRNAs from degradation and harbors molecules that allow specific targeting to recipient cells. Although several studies have investigated the miRNA content in milk EVs from individual species, little is known about the evolutionary conserved nature of EV-associated miRNAs among different species. In this study, we profiled the miRNA content of purified EVs from human and porcine milk. These data were compared to published studies on EVs from human, cow, porcine, and panda milk to assess the overlap in the top 20 most abundant miRNAs. Interestingly, several abundant miRNAs were shared between species (e.g., let-7 family members let-7a, let-7b, let-7f, and miR-148a). Moreover, these miRNAs have been implicated in immune-related functions and regulation of cell growth and signal transduction. The conservation of these miRNA among species, not only in their sequence homology, but also in their incorporation in milk EVs of several species, suggests that they are evolutionarily selected to regulate cell function in the newborn.

Arrest of in vivo growth of a solid Leydig cell tumor by prolonged inhibition of mitochondrial protein synthesis.

Oxytetracycline was given by means of chronic i.v. infusion, in amounts which impair specifically mitochondrial protein synthesis, to Wistar rats carrying a solid Leydig cell tumor. The prolonged inhibition of mitochondrial protein synthesis finally results in proliferation arrest of the s.c. growing tumor. As long as the tumor is growing, the energy-generating capacity of the mitochondrial becomes diluted, until it is reduced to a critical level, which results in growth arrest. This cytostatic effect is found after treatment for 8 to 12 days. During this period, the tumor volume increases to an extent comparable to 2 to 3 tumor cell divisions. The proliferation arrest found is, at least after treatment for 3 weeks, reversible. Withdrawal of oxytetracycline results in continuation of tumor growth after a latent period of about 5 days. The data confirm previous observations made in experimental systems about the usefulness of mitochondrial protein synthesis as target for cancer chemotherapy. They support, moreover, the explanation given for the marked prolongation of survival time found in tetracycline-treated patients with a squamous cell carcinoma of the larynx-pharynx. If antineoplastic therapy, based on inhibition of mitochondrial protein synthesis, is considered, the tetracyclines should be the drugs of choice, because their toxic side effects are minimal at dosages which cause tumor proliferation arrest.

Doxycycline in combination chemotherapy of a rat leukemia.

Inhibition of mitochondrial protein synthesis by doxycycline (DC), a tetracycline analogue, has significant antitumor effects in several tumor systems. In the present study, the effects of continuous DC treatment combined with intermittent administration of Adriamycin or 1-beta-D-arabinofuranosyl cytosine on the growth of a rat leukemia were investigated. The presence of DC retards tumor relapse after 1-beta-D-arabinofuranosyl cytosine or Adriamycin treatment significantly. DC may therefore be of value in several modalities of antitumor treatment.

Arrest of in vivo proliferation of Zajdela tumor cells by inhibition of mitochondrial protein synthesis.

The proliferation of Zajdela tumor cells, grown in vivo in Wistar rats, is arrested by low amounts of oxytetracycline. Oxytetracycline is administered by means of continuous infusion in such a way that the oxytetracycline concentration in serum and ascitic fluid is maintained at a level at which only mitochondrial protein synthesis is blocked. Under these conditions, Zajdela mitochondrial tumor cells cease dividing after a few cell generations, an event which is preceded by reduction of cytochrome c oxidase activity of the tumor cells. Toxicity to host tissues is limited to the immune system which is apparently suppressed by the drug. Even so, oxytetracycline might have therapeutic potential in human cancer therapy, especially because it does not influence the hemopoietic system.

Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis.

The results described in this paper demonstrate that proliferation arrest by low concentrations of tetracyclines, which has previously been shown in experiments with animal tumor systems, can also be achieved in tumor systems of human origin. Tetracyclines specifically inhibit mitochondrial protein synthesis. Prolonged and continuous impairment of protein synthesis inside the mitochondria leads to reduction of the cellular concentration of the polypeptide products which are coded and synthesized within mitochondria. These products are part of the oxidative phosphorylative system of the cell. Long-term tetracycline treatment leads to a decrease of oxidative ATP-generating capacity as monitored by cytochrome c oxidase activity. This may cause severe energetic or metabolic disturbances which explain the proliferation arrest observed. Proliferation arrest, provided that mitochondrial protein synthesis is blocked effectively, is found in vitro as well as in vivo. It is shown that the effect of doxycycline is not limited to cytostasis; prolonged doxycycline treatment is clearly cytotoxic for the tumor cells.

Mitochondrial DNA from various organisms does not contain internally methylated cytosine in -CCGG- sequences.

Mitochondrial DNAs from yeast, Neurospora, rat and calf do not contain internally methylated cytosine in -CCGG- sequences.

The ribosomal RNA genes on Neurospora crassa mitochondrial DNA are adjacent.

Hybridization of separated 24 S and 17 S ribosomal RNA from Neurospora crassa mitochondrial ribosomes to restriction fragments of mitochondrial DNA leads to the conclusion that the large and small ribosomal RNA are adjacent on the restriction endonuclease cleavage map of the DNA. The distance between the two genes is estimated at 900 basepairs. This result is consistent with the existence of a ribosomal precursor RNA in N. crassa mitochondria and is in contrast to the situation in yeast, where the ribosomal genes are far apart on the mitochondrial DNA. The position of the ribosomal RNA genes on the cleavage map of N. crassa mtDNA provides a start for ordering the Hind III restriction fragments.

The biogenesis of rat liver mitochondrial ATPase. Subunit composition of the normal ATPase complex and of the deficient complex formed when mitochondrial protein synthesis is blocked.

1. An ATPase complex containing 12 subunits was isoalted from rat liver mitochondria. 2. In vivo inhibition of mitochondrial protein synthesis by the chloramphenicol analogue thiamphenicol leads to the formation of an oligomycin-insensitive membrane-bound ATPase complex in mitochondria of regenerating rat liver. 3. This oligomycin-insensitive, membrane-bound ATPase was isolated by the same procedure as the ATPase complex from regenerating livers of untreated animals. 4. SDS-polyacrylamide gel electrophoresis of in vivo labelled ATPase complexes from control and from thiamphenicol-treated rats reveals that three subunits out of the 12 are not synthesized or assembled when the mitochondrial translation activity is blocked. 5. From the subunits synthesized and assembled when mitochondrial pror (Fo) of the ATPase complex (subunit 5). 6. The oligomycin sensitivity-conferring protein seems absent in the ATPase complex formed in the presence of thiamphenicol.

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria.

1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.

The biogenesis of rat mitochondrial ATPase. Two subunits are synthesized inside the mitochondria.

Isolated mitochondria from regenerating rat liver synthesize at least five different polypeptides with molecular weights ranging from 19 000 to 43 000. Among these, two polypeptides with molecular weights of 22 000 and 25 ooo could be identified as ATPase subunits. It has previously been shown that these subunits, designated 6 and 7, are lacking in the ATPase complex that is formed in vivo when mitochondrial protein synthesis is blocked by thiamphenicol treatment. The 22 000 Mr protein is enriched in the fraction containing the fully assembled ATPase complex, whereas the 25 000 Mr protein is not.

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa.

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21-26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.

The restriction endonuclease cleavage map of rat liver mitochondrial DNA.

Mitochondrial DNA from rat liver contains six sites for cleavage by the restriction endonucleases Hind III and EcoRI. A large stretch of DNA, comprising about 40% of the mitochondrial genome is not cleaved by either of the enzymes; eight cleavage sites are located on a DNA stretch of 35% of the genome length suggestive of an unequal distribution of the A - T baspairs over the molecule. The number of Hind III and Eco R I fragments is much higher than reported for other mammalian mitochondrial DNAs up to now.

The heterogeneity of rat-liver mitochondrial DNA.

Two types of mitochondrial DNA (mtDNA) can be distinquished in an inbred strain of rats of the Wistar type. The population of DNA molecules of the liver of one single rat is homogeneous. This was shown for a number of 100 animals and confirms the data of other investigators. The two types of mitochondrial DNA, designated A and B, differ in their number of cleavage sites for the restriction endonucleases Eco RI (2sites), Hind II (1 site) and Hha I (1 site). No differences were found for the restriction enzymes Bam HI, Hap II, Hind III and Hpa I. The degree of sequence divergence of the two types of DNA is calculated to be roughly 5% on the basis of these observations. From 20 rats part of the liver was taken and the mtDNA was characterized. Heterologous and homologous crosses between type A and type B rats were made. Analysis of the offspring revealed strictly maternal inheritance of the A and B mtDNA traits. For purposes of base-sequence analysis and RNA.DNA hybridization the strain could easily be "purified" genetically.

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline.

Under standard conditions, liver regeneration is impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 micrograms/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 micrograms/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.

Further analysis of the type differences of rat-liver mitochondrial DNA.

1. The sequence of the small Hind III fragment F of rat-liver mitochondrial DNA (mtDNA) type A and type B was determined in order to investigate the nature of the differences between the two types of mtDNA and to determine its position in the Hind III fragment map. 2. The three differences found were point mutations. No deletion or insertion and no modification was observed. Two of the three differences affect the sequences which are recognition sites for Eco RI, Alu I and Taq I in type A, but not in type B, mtDNA. 3. The presence of an Eco RI restriction site only within the Hind III fragment F of type A shows that the fragment is situated in between the Hind III fragments A and E. 4. In one of the six reading frames, the Hind III fragment F contains the code for a carboxyl-terminal end of a polypeptide in which the three mutations do not lead to alterations in the possible aminoacid sequence. 5. The restriction sites for Taq I and a number of the sites for Alu I and Hae III were mapped. 6. The positions of the Hap II fragment J, and of a Hind III fragment G on the mtDNA were determined.

Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells.

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.

Mitochondrial biogenesis during the activation of lymphocytes by mitogens: the immunosuppressive action of tetracyclines.

The role of mitochondrial biogenesis and function during mitogenic stimulation of rat thymocytes was investigated. The results show that mitochondrial biogenesis is required to provide the ATP for the energy-requiring processes occurring during blastogenesis. Impairment of mitochondrial biogenesis by inhibition of mitochondrial protein synthesis inhibits blast transformation. Since the tetracyclines impair mitochondrial protein synthesis, the results offer an explanation for the well-known immunosuppressive effects of these antibiotics.

Specific inhibition of mitochondrial protein synthesis influences the amount of complex I in mitochondria of rat liver and Neurospora crassa directly.

Specific inhibition of mitochondrial protein synthesis reduces the oxidation rate of NADH-linked substrates in rat liver as well as in Neurospora crassa mitochondria. The present study shows that this is due to the fact that inhibition of mitochondrial protein synthesis leads to a decrease of the concentration of active complex I. Therefore, these results demonstrate that at least one of the genes for the subunits of complex I is localized on mitochondrial DNA.