Review of the scientific basis for establishing the essentiality of trace elements

The recent Expert Consultation of World Health Organization (WHO)/Food and Agricultural Organization (FAO)/International Atomic Energy Agency (IAEA) defined essentiality of a trace element as follows: "An element is considered essential to an organism when reduction of its exposure below a certain limit results consistently in a reduction in a physiologically important function, or when the element is an integral part of an organic structure performing a vital function in the organism." This definition omits a previous postulate that the mechanism of action of an essential trace element should be well defined; it also supersedes another criterion, once suggested for essentiality, a normal, rather than log-normal distribution of an element's tissue concentrations. The Expert Consultation offers no generally applicable criteria for the physiological importance of functions, and that determination is left to expert groups charged with setting national and other nutritional recommendations. The use of the term "physiological" rather than "biochemical" strongly implies that neither changes of an element's concentration nor of a specific enzyme function alone are proof of essentiality. Among physiologically important functions are growth, reproduction, longevity, and all metabolic and hormonal functions that bear a clear, inverse relation to disease risk. Finally, the term "consistent" states the need for independent confirmation of the original data, before an element can be recognized as essential. These definitions will be discussed as background for further discussions of our present knowledge of boron (B).     

The evolutionarily conserved ribosomal protein L23 and the cationic urease beta-subunit of Yersinia enterocolitica O:3 belong to the immunodominant antigens in Yersinia-triggered reactive arthritis: implications for autoimmunity.

BACKGROUND: Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). MATERIALS AND METHODS: Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. RESULTS: Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease beta-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease beta-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. CONCLUSION: Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.     

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata

We have determined the complete nucleotide sequence of the small- subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.      

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Leader-encoded open reading frames modulate both the absolute and relative rates of synthesis of the virion proteins of simian virus 40.

Numerous viral and cellular RNAs are polycistronic, including several of the late mRNA species encoded by simian virus 40 (SV40). The functionally bicistronic major late 16S and functionally tricistronic major late 19S mRNA species of SV40 contain the leader-encoded open reading frames (ORFs) LP1, located upstream of the sequence encoding the virion protein VP1, and LP1*, located upstream of the sequence encoding the virion proteins VP2 and VP3. To determine how these leader ORFs affect synthesis of the virion proteins, monkey cells were transfected with viral mutants in which either the leader-encoded translation initiation signal was mutated or the length and overlap of the leader ORF relative to the ORFs encoding the virion proteins were altered. The levels of initiation at and leaky scanning past each initiation signal were determined directly by quantitative analysis of the viral proteins synthesized in cells transfected with these mutants. Novel findings from these experiments included the following. (i) At least one-third of ribosomes bypass the leader-encoded translation initiation signal, GCCAUGG, on the SV40 major late 16S mRNA. (ii) At least 20% of ribosomes bypass even the consensus translation initiation signal, ACCAUGG, when it is situated 10 bases from the 5' end on the major late 16S mRNA. (iii)O The presence of the leader ORF on the bicistronic 16S mRNA species reduces VP1 synthesis threefold relative to synthesis from a similar RNA that lacks it. (iv) At least half and possibly all VP1 synthesized from the bicistronic 16S mRNA species is made by a leaky scanning mechanism. (v) LP1 and VP1 are synthesized from the bicistronic 16S mRNA species at approximately equal molar ratios. (vi) Approximately half of the VP1 synthesized in SV40-infected cells is synthesized from the minor, monocistronic 16S mRNA even though it accounts for only 20% of the 16S mRNA present. (vii) The presence and site of termination of translation of the leader ORF on the late 19S mRNAs affect the relative as well as absolute rates of synthesis of VP2 and VP3.     

Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

A Reversible Histone H3 Acetylation Cooperates with Mismatch Repair and Replicative Polymerases in Maintaining Genome Stability

Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.