Constant copy numbers of plasmids inSaccharomyces cerevisiae hosts with different ploidies

Summary The cellular -galactosidase activities produced by thelac'Z gene ofEscherichia coli, cloned on YEp, YRp, or YCp-type plasmids in host cells ofSaccharomyces cerevisiae with different ploidies, and which was expressed by a modified jeastHIS5 promoter, showed characteristic differences depending on the plasmid. But for any given plasmid, the isogenic diploid and tetraploid transformants showed slightly lower enzyme activities than their respective haploid transformants. This was due to the similar copy numbers of the plasmids in host cells. Since the cell number per unit volume of the culture decreased with increasing cell ploidy, the enzyme activity per unit volume of the culture decreased significantly. The holding stability of plasmids increased with increasing ploidy of the host cell, especially that of the YRp plasmid. On the YRp plasmid, thelac'Z gene showed higher productivity withTRP1 thanLEU2 as the selection marker for the plasmid.     

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).     

Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies

Abstract We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyl-transferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans . The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.     

Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

Background

Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies.

Methodology/Principal Findings

Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b⁺/CD11c⁺ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines.

Conclusions/Significance

The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy.     

Tissue-specific interactions of TNI isoforms with other TN subunits and tropomyosins in C. elegans: the role of the C- and N-terminal extensions

The aim of this study is to investigate the function of the C-terminal extension of three troponin I isoforms, that are unique to the body wall muscles of Caenorhabditis elegans and to understand the molecular interactions within the TN complex between troponin I with troponin C/T, and tropomyosin. We constructed several expression vectors to generate recombinant proteins of three body wall and one pharyngeal troponin I isoforms in Escherichia coli. Protein overlay assays and Western blot analyses were performed using antibodies. We demonstrated that pharyngeal TNI-4 interacted with only the pharyngeal isoforms of troponin C/T and tropomyosin. In contrast, the body wall TNI-2 bound both the body wall and pharyngeal isoforms of these components. Similar to other invertebrates, the N-terminus of troponin I contributes to interactions with troponin C. Full-length troponin I was essential for interactions with tropomyosin isoforms. Deletion of the C-terminal extension had no direct effect on the binding of the body wall troponin I to other muscle thin filament troponin C/T and tropomyosin isoforms.     

Long-term effects of peginterferon alfa-2a therapy in Japanese patients with chronic hepatitis B virus infection

Background

There is no information on the long-term effects of peginterferon (PEG-IFN) alfa-2a therapy for chronic hepatitis B (CHB) in Japan. This double-blind, randomized trial investigated the efficacy of PEG-IFN therapy.

Methods

We analyzed 22 Japanese patients with CHB (hepatitis B e antigen [HBeAg]-positive: 17, HBeAg-negative: 5) treated with PEG-IFN alfa-2a and followed-up posttreatment for 5 years. Responders represented patients who showed persistent normalization of alanine transferase (ALT) levels, HBeAg clearance, and low hepatitis B virus (HBV) DNA levels (HBeAg-positive patient; <5 log copies/mL, HBeAg-negative patient; <4.3 log copies/mL) at end of treatment, and at 1, 2, 3, 4 and 5 years posttreatment. In addition, baseline HBeAg-positive patients who showed sustained normalization of ALT level, HBeAg clearance, and low HBV DNA level for more than 6 months until at 1, 2, 3, 4, and 5 years after completion of PEG-IFN were also classified as “triple responders” and the proportion of triple responders relative to all patients was termed the “triple response rate”.

Results

The response rates among HBeAg-positive patients were 13 %, 25 %, 14 %, 21 % and 21 % at end of treatment, and at 1, 2, 3, 4, and 5 years, respectively. The response rate tended to be higher in patients treated for 48 than 24 weeks. The respective response rates among HBeAg-negative patients were 0 %, 20 %, 20 %, 20 % and 25 %. During the treatment period, hepatitis B surface antigen (HBsAg) clearance at 3.5 years was noted in one patient, who was 37-year-old, male, had genotype C and received PEG-IFN alfa-2a at 90 μg for 48 weeks.

Conclusion

At 5 years after completion of PEG-IFN, the triple response rate in HBeAg-positive patients and combined response rate in HBeAg-negative patients were 21 % (3/14) and 25 % (1/4), respectively. The triple response was seen in three patients who had all been treated with PEG-IFN for 48 weeks.

     

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Summary The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, of R6K or protein RepH encoded by and iniating the replication of pC194.