Identification of important bases for the self-cleavage activity at two single-stranded regions of genomic HDV ribozyme.

In order to determine important bases at two single-stranded regions [SSrA (726-731 nt) and SSrB (762-766)] derived mainly from secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several point mutants at these two regions on the HDV88 molecule (683-770). Among the bases at SSrA and SSrB regions C763 was found to play an essential role during self-cleavage process since substitutions to any other bases viz. A or G or U completely abolished the activity.     

Enzymatic synthesis of chimeric tRNAs with unusual numbers of base pairs in the anticodon stem; their structure and properties

Chimeric tRNATyr molecules have been constructed by enzymatic procedures in vitro from the 5'-half fragment of T. utilis tRNATyr and the 3'-half fragment of yeast tRNATyr, and vice versa. These chimeric tRNAs contain base-mismatching(s) in the anticodon stem and, therefore, have only 3 or 4 base pairs in the stem. Although the Tm of these chimeras are largely decreased, there seems to be no gross difference between the structure of native and chimeric tRNATyrs at physiological temperatures in the presence of 10 mM MgCl2. Aminoacylation assays also revealed that the tyrosine-acceptance of the chimeras are fully comparable to that of native tRNATyrs. However, the possibility remains that the properties of the chimeras are considerably different from those of native ones at lower Mg++ concentrations.     

A note on colony-blot double-stain method for isolation of Vibrio cholerae serotype 01 from specimens heavily contaminated with non-01 Vibrio cholerae

A colony-blot double-stain method was developed to identify individual colonies of Vibrio cholerae serotype 01 (pandemic strain) in mixed bacterial cultures on solid media. The colonies are transferred from agar to nitrocellulose membranes for an enzyme-linked immunosorbent assay (ELISA). Colonies of 01 vibrios bind the enzyme-linked antibodies and appear as brown dots on the membranes; pale black dots develop at the site of replicated colonies of other bacteria as a result of the activity of endogenous oxidase-like enzymes and serve as reference points. The results indicate that the colony-blot double-stain method is useful for the isolation of colonies of V. cholerae serotype 01 in specimens that are heavily contaminated with non-01 vibrios.     

Detecting amplicons of loop‐mediated isothermal amplification

Loop‐mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real‐time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real‐time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.     

In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

Localization of adherens junction proteins along the possible sliding interface between secretory ameloblasts of the rat incisor

Localization of junctions between inner enamel-secretory ameloblasts was examined by immunofluorescence microscopy using antibodies against adherens junction proteins, radixin, vinculin, and A-CAM. All antibodies used stained the boundary between the ameloblasts exclusively in the plane where F-actin was abundant. This suggests that the adherens junctions in the ameloblasts are involved in cell-to-cell movement with actin-based microfilament bundles.     

The molecular basis of mutations at the Waxy locus from Amaranthus caudatus L.: evolution of the waxy phenotype in three species of grain amaranth

Polymorphisms at the Waxy locus of Amaranthus caudatus L. collected from a wide range of regions were used to investigate genetic diversity and mutation sites. A comparison of the Waxy locus revealed a very high level of sequence conservation. This result clearly showed low environmental and evolutionary variability in the Waxy gene. We also performed screening to confirm the mutation sites in the coding sequences of all accessions. The results indicate that one insertion in the coding region of Waxy genes was responsible for the change in perisperm starch leading to the waxy phenotype in all accessions of this species, and thus that a single mutation event altered the regulation of the Waxy gene during the domestication of this crop. In addition, phylogenetic analysis showed that waxy phenotypes within each of three species, A. caudatus, A. cruentus and A. hypochondriacus, originated separately or differentiated from nonwaxy phenotypes of each species through a single mutational event (i.e., a frame shift or base substitution). We also compared obvious structural features of the coding sequence of waxy and nonwaxy phenotypes with those of low-amylose phenotypes in A. caudatus. The Waxy coding sequences of low-amylose phenotypes do not show polymorphisms and are identical with those of waxy phenotypes. This could mean that there is another gene that encodes a key enzyme responsible for amylose synthesis as the elementary quantity in tissues other than perisperm in A. caudatus.