Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.     

Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.     

Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.

We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.     

The hydroxypyridinium crosslinks of skeletal collagens: Their measurement,properties and a proposed pathway of formation

A method is described for quantifying both reducible and mature crosslinking amino acids of collagen. The main crosslinking residue in cartilage, dentine and mature bone was the 3-hydroxypyridinium compound identified by Fujimoto et al. (1–3). Adult articular cartilage contained about one residue per collagen molecule, over forty times the content of the reducible crosslinks. We propose that hydroxypyridinium residues are formed by spontaneous interaction of two residues of hydroxylysino-5-ketonorleucine. This reaction explains the disappearance of reducible crosslinks at maturity and provides a novel mechanism for lateral crosslinking within and between fibrils which may account for some of the unique physical properties of hard tissue collagens.     

Effects of Parenteral Lipid Infusion on DNA Damage in Very Low Birth Weight Infants

Very low birth weight (VLBW) infants are known to have poorly developed antioxidant system and may be at increased risk for radical damage. Previous studies have reported higher levels of lipid peroxide products in lipid emulsion used for parenteral nutrition. To examine the direct effects of parenteral lipid infusion on DNA damage in VLBW infants, we measured urinary 8-hydroxydeoxyguanosine (8-OHdG) levels in VLBW infants before, during, and after the parenteral lipid infusion. In both the lipid-infused and lipid-free groups, urinary 8-OHdG excretion levels at 14 days old were significantly ( p <0.01) lower than those at 2 and 7 days old. However, there were no significant differences in urinary 8-OHdG excretion levels between the lipid-infused and lipid-free groups at 2, 7, and 14 days old. Our results suggest that parenteral lipid infusion does not cause oxidative DNA damage, but irrespective of the infusion DNA damage during the first week of life is enhanced when compared with 14 days after birth in VLBW infants.     

Trophic position and growth stages of Caddisfly (Stenopsyche marmorata Navas) larvae in the Echi River, Japan

We investigated the trophic position of the larval filter-feeding caddisfly species—Stenopsyche marmorata Navas in the Echi River (Japan) using δ¹³C and δ¹⁵N measurements by instar growth stage. We also analyzed δ¹³C and δ¹⁵N of food sources (periphyton and suspended and benthic POM) along the course of the river. The larvae showed some degree of spatial variation in δ¹³C and δ¹⁵N values with a downstream increase in δ¹³C and δ¹⁵N values. Our results demonstrate that the trophic structure of S. marmorata of different larval stages were spatially varying, depending on food sources. Small-sized instars displayed a higher trophic position than larger instars, confirming that their non-selective feeding behavior leads to consumption of items with high δ¹⁵N values according to the spatial distribution of food sources in the riverine system.     

Insights into β-Lactamases from Burkholderia Species,Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k_cat values ranging from 0.38 ± 0.04 to 460 ± 46 s⁻¹ and possesses high k_cat/k_inact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest k_cat value for cefotaxime of 9.0 ± 0.9 s⁻¹. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k_cat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.     

The relationship between consumption of tyrosine and phenylalanine as precursors of catecholamine at breakfast and the circadian typology and mental health in Japanese infants aged 2 to 5 years

Background

This study aims to examine the relationship between tyrosine and phenylalanine intake at breakfast as precursors of dopamine, and scores on the Torsvall-Åkerstedt Diurnal Type Scale and of mental health in Japanese infants aged 2 to 5 years.

Results

An integrated questionnaire was administered to parents of 1,367 infants attending one of ten nursery schools governed by Kochi City or a kindergarten affiliated with the Faculty of Education at Kochi University (775 answers for analysis: 56.7%) in May and June 2008. Questionnaires included the Torsvall-Åkerstedt Diurnal Type Scale and questions on sleep habits (onset, offset, quality, quantity, and so on), meal habits (content and regularity of timing), and mental health (depressive states). Amount of tyrosine and phenylalanine intake was calculated based on a breakfast content questionnaire and data on the components of amino acids in foods. Infants who ingested more than 800 mg of tyrosine or phenylalanine at breakfast per meal were more morning-type than those who ingested less than 800 mg (ANOVA: P= 0.005). However, this relationship disappeared in the ANCOVA analysis (with the covariance of tryptophan intake, P= 0.894). Infants who ingested more than 800 mg of the two amino acids at breakfast showed significantly higher mental health scores (lower frequency of depressive states) than those who ingested less than 800 mg (ANOVA: P = 0.004). This relationship remained significant when ANCOVA analysis was performed with the covariance of tryptophan (ANCOVA: P= 0.017).

Conclusions

These results suggest that tyrosine and phenylalanine ingested at breakfast are not related with circadian phase, but are relate with mental health in infants.