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.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

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.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

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.