Isolation of an Insertion Sequence from Ralstonia solanacearum Race 1 and Its Potential Use for Strain Characterization and Detection

A new insertion sequence (IS), IS1405, was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS5 family, but the extent of nucleotide sequence identity in 5′ and 3′ noncoding regions between IS1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS1405 and additional five endogenous IS1405s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.     

Regulation of Hedgehog signaling by ubiquitination

The Hedgehog (Hh) signaling pathway plays crucial roles both in embryonic development and in adult stem cell function. The timing, duration and location of Hh signaling activity need to be tightly controlled. Abnormalities of Hh signal transduction lead to birth defects or malignant tumors. Recent data point to ubiquitination-related posttranslational modifications of several key Hh pathway components as an important mechanism of regulation of the Hh pathway. Here we review how ubiquitination regulates the localization, stability and activity of the key Hh signaling components.     

Evaluation of EGFR-targeted radioimmuno-gold-nanoparticles as a theranostic agent in a tumor animal model

This study evaluated the tumor targeting and therapeutic efficacy of a novel theranostic agent ¹³¹I-labeled immuno-gold-nanoparticle (¹³¹I-C225-AuNPs-PEG) for high epidermal growth factor receptor (EGFR)-expressed A549 human lung cancer. Confocal microscopy demonstrated the specific uptake of C225-AuNPs-PEG in A549 cells. ¹³¹I-C225-AuNPs-PEG induced a significant reduction in cell viability, which was not observed when incubated with AuNPs-PEG and C225-AuNPs-PEG. MicroSPECT/CT imaging of tumor-bearing mice after intravenous injection of ¹²³I-C225-AuNPs-PEG revealed significant radioactivity retention in tumor suggested that ¹³¹I-labeled C225-conjugated radioimmuno-gold-nanoparticles may provide a new approach of targeted imaging and therapy towards high EGFR-expressed cancers.     

Frequency of glucose-6-phosphate dehydrogenase (G6PD) mutations in Chinese,Filipinos, and Laotians from Hawaii

In a Hawaii Hereditary Anemia Screening Project, 4,984 participants were tested for glucose-6-phosphate dehydrogenase (G6PD) deficiency by a filter paper blood spot fluorescence test. Abnormal samples and suspected heterozygotes were checked by quantitative G6PD assay (normal 4.5 to 14 units/g Hb). G6PD was deficient (< 1.5 units/g Hb) in 188 of 2,155 males; 7 other males had low activity (1.5 to 2.8 units/g Hb). The gene frequency, estimated from males after excluding referred and related cases, was 0.037 for Chinese, 0.134 for Filipinos, and 0.203 for Laotians. Among 2,829 females tested, family data showed 111 females were obliged to be at least heterozygous, regardless of G6PD activity, and 43 others had low G6PD activity. Most heterozygotes probably remained undetected by G6PD screening. In 28 females, activity was under 10%; in another 9 females, activity was < 1.5 units/g Hb. Since only 25 homozygotes would be predicted, this apparent excess of females with deficient activity could be due to unequal X-inactivation in some heterozygotes. DNA analysis by polymerase chain reaction amplification and special analytic procedures revealed 10 different missense mutations in 75 males. The nucleotide 835 AT and 1360 CT transitions were first detected in this Hawaiian Project; we found that the nucleotide 1360 mutation was the most common cause of G6PD deficiency in Filipinos. This is the first report of G6PD screening and analysis of molecular G6PD mutations in Filipino and Laotian populations.     

The effects of electrophoretically "slow" and "fast" alpha-2 macroglobulin on mixed lymphocyte cultures

It has been determined that alpha-2 macroglobulin (alpha 2M) is more suppressive of a mixed lymphocyte response (MLR) when complexed with proteinase than in its "native" state. Other alpha 2M preparations showed a moderate level of MLR suppression, but it is unlikely that this is a result of interaction with cellular proteinases. A panel of other proteinase inhibitors (alpha 1 PI, SBTI, BPTI, TLCK) did not suppress the MLR to the same extent as alpha 2M either when bound with or free from trypsin. A dose-responsive pattern of MLR suppression similar to that observed with purified proteinase-complexed alpha 2M was seen with serum containing proteinase-complexed alpha 2M. The population of cells that apparently conveys the suppressive property is the adherent cells (putative monocytes), which can reduce the MLR almost as well as unfractionated cells when exposed to alpha 2M. Most of these properties of alpha 2M were demonstrable in "serumless" medium with qualitative similarity to the MLR obtained in cultures performed with conventional serum supplemented medium. It was found that alpha 2M-trypsin complexes must be presented at or near culture initiation and remain in contact with the cells for a minimum of approximately 4 hr to have its optimum effect.     

Genetics of propionic acidemia in a Mennonite-Amish kindred.

A large Mennonite kindred was found to have propionic acidemia (complementation group pcc C) in at least four different sibships. Even within this kindred and this complementation group (where etiology may be assumed to be identical), there is a wide range of symptoms exhibited by homozygous pcc C-deficient individuals. The inbreeding coefficients (f) for the affected sibships ranged from 4.776 X 10(3) to 2.003 X 10(-2). Data from this study strongly support the single-locus autosomal recessive mode of inheritance. Three couples were found to be common in the ancestry (9--11 generations ago) of all eight parents of the four affected sibships. Relative likelihoods for a member of each of those couples to have been the early carrier of the defective allele were calculated at 1539, 278, and 1. Thus, one couple was designated the most likely earliest-known transmitter of the pcc-deficient allele.     

alpha-fetoprotein binding specificity for arachidonate, bilirubin, docosahexaenoate, and palmitate. A spin label study

A dianionic spin label, 1-L-glutamate-5-N-(1-oxyl-2,2,6,6-tetramethyl-4-aminopiperidinyl)-i,4-dinitrobenzene, has been used to probe the relative binding specificity of a single anionic ligand site on bovine alpha-fetoprotein (AFP) to arachidonate, bilirubin, docosahexaenoate, and plamitate. The binding isotherm of the spin label with AFP, as shown by a Scatchard plot, indicates the presence of a single high affinity binding site. The site-site relationship of the four endogenous ligands, arachidonate, bilirubin, docosahexaenoate, and palmitate, was determined by studying their effectiveness in competing for this anionic ligand binding site on AFP. Scatchard plots of the spin label in the presence of 1 to 3 molar equivalents of arachidonate, bilirubin, and docosahexaenoate and up to 6 molar equivalents of palmitate have been determined. The effectiveness of the four endogenous ligands in displacing the spin label from its primary binding site is bilirubin greater than or equal arachidonate approximately equal to docosahexaenoate greater than palmitate. These results indicate that polyunsaturated essential fatty acids and bilirubin share a high affinity binding site on AFP. We propose that the function of this anionic ligand binding site on AFP is for the transport of bilirubin and polyunsaturated fatty acids in fetal serum, as well as for the cross-placental transfer of this metabolite and of essential fatty acids.