Isolation of intact Sm/RNP antigens from rabbit thymus

A comparison of the immunologically reactive components of the highly conserved Sm and RNP autoantigens from various mammalian tissue sources suggested the complete absence of a major 26K to 27K Sm-specific polypeptide in rabbit thymus extracts prepared by conventional procedures. A simple modification of the solubilization protocol, achieved by sonicating a suspension of commercial rabbit thymus acetone powder in 0.35 M NaCI, gave an extract containing the full complement of immunologically reactive Sm and RNP proteins detectable in other mammalian species. Without further manipulation, extracts were immediately passed through an immunoaffinity column constructed from human SLE IgG with both anti-Sm and anti-RNP reactivities. The proteins of the purified Sm/RNP were recovered in sufficient quantities for direct analysis by protein staining or immunoblot assays. The antigenic polypeptides were recovered intact and consisted of a single 73K RNP-specific species together with Sm-specific proteins of 26K to 27K (a doublet) and 13K. These proteins were easily visible by protein stain as were nonantigenic components of 35K, 32K, 11K, and less than 10K. The same polypeptides were present in affinity-purified Sm/RNP from HeLa cells, although the RNP protein was slightly smaller. The resolution and integrity of the complexes isolated by this simple two-step procedure, requiring less than 4 hr for completion, is remarkable, and the protein composition of the product compares quite favorably with antigens isolated from other sources by considerably more lengthy and laborious procedures.     

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.

Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Future directions for root caries research

Interest among researchers in the diagnosis, aetiology, prevention, and treatment of root caries has increased substantially over the past two decades. However, there are some fundamental problems impeding the advancement of the field which remain to be addressed and resolved. A universally acceptable definition of root caries is not yet available. The relationship of root caries to coronal caries has not been established. The underlying disease process is still not clearly understood. The optimal utilisation of preventive/therapeutic agents for the treatment or prevention of root caries has not been determined. New treatment materials and preventive agents have not yet been tested in controlled clinical trials. These are a few of the issues and problems which we address in this paper.     

New techniques for physical mapping of the human genome.

We describe improvements in techniques and strategies used for making maps of the human genome. The methods currently used are changing and evolving rapidly. Today's techniques can produce ordered arrays of DNA fragments and overlapping sets of DNA clones covering extensive genomic regions, but they are relatively slow and tedious. Methods under development will speed the process considerably. New developments include a range of applications of the polymerase chain reaction, enhanced procedures for high resolution in situ hybridization, and improved methods for generating, manipulating, and cloning large DNA fragments. More detailed genetic and physical maps will be useful for finding genes, including those associated with human diseases, long before the complete DNA sequence of the human genome is available.     

The role of methionine in regulating folate-dependent reactions in isolated rat hepatocytes

In isolated rat hepatocytes, histidine and formate, are oxidized to CO₂ by folate-dependent reactions. These reactions are stimulated two- to fourfold by the addition of l-methionine, dl-homocysteine, S-adenosyl-l-methionine (Ado-Met), or S-adenosyl-l-homocysteine (Ado-Hcy). These compounds all increase the hepatocyte concentration of Ado-Met and Ado-Hcy. Substrates of hepatic catechol O-methyltransferase, such as l-Dopa methyl ester and 3,4-dihydroxyphenylacetic acid, decrease the hepatocyte concentration of Ado-Met in the presence or absence of added l-methionine or dl-homocysteine. The catechols do not affect the concentration of Ado-Hcy, but they inhibit the oxidation of formate and histidine. Thus, there is an excellent positive correlation between the rate of histidine and formate oxidation and the concentration of Ado-Met. There is no correlation between the rate of these reactions and either the Ado-Hcy concentration or the concentration ratio of Ado-Met:Ado-Hcy. Ado-Met inhibition of rat hepatic 5,10-methylene tetrahydrofolate reductase activity is reversed by Ado-Hcy, but the dependency of rat hepatic 5-methyltetrahydrofolate-homocysteine transmethylase activity (methionine synthetase) on Ado-Met is not altered by Ado-Hcy. These results indicate that methionine, through its conversion to Ado-Met, regulates folate-dependent reactions in isolated hepatocytes by increasing activity of methionine synthetase which leads to an increased concentration of tetrahydrofolate. That methionine and Ado-Met increase the hepatocyte concentration of nonmethyltetrahydrofolate compounds and decrease the hepatocyte concentration of 5-methyltetrahydrofolate supports this hypothesis.     

Psychological processes mediating the association between developmental trauma and specific psychotic symptoms in adults: a systematic review and meta‐analysis

Experiencing psychological trauma during childhood and/or adolescence is associated with an increased risk of psychosis in adulthood. However, we lack a clear knowledge of how developmental trauma induces vulnerability to psychotic symptoms. Understanding the psychological processes involved in this association is crucial to the development of preventive interventions and improved treatments. We sought to systematically review the literature and combine findings using meta‐analytic techniques to establish the potential roles of psychological processes in the associations between developmental trauma and specific psychotic experiences (i.e., hallucinations, delusions and paranoia). Twenty‐two studies met our inclusion criteria. We found mediating roles of dissociation, emotional dysregulation and post‐traumatic stress disorder (PTSD) symptoms (avoidance, numbing and hyperarousal) between developmental trauma and hallucinations. There was also evidence of a mediating role of negative schemata, i.e. mental constructs of meanings, between developmental trauma and delusions as well as paranoia. Many studies to date have been of poor quality, and the field is limited by mostly cross‐sectional research. Our findings suggest that there may be distinct psy­chological pathways from developmental trauma to psychotic phenomena in adulthood. Clinicians should carefully ask people with psychosis about their history of developmental trauma, and screen patients with such a history for dissociation, emotional dysregulation and PTSD symptoms. Well conducted research with prospective designs, including neurocognitive assessment, is required in order to fully understand the biopsychosocial mechanisms underlying the association between developmental trauma and psychosis.