Isolation and characterization of arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii

Arsenate-sensitive and resistant mutants of Chlamydomonas reinhardtii were obtained by screening mutants generated by random insertional mutagenesis for growth in the presence of various concentrations of arsenate. The intracellular concentrations of arsenic in the mutants kept in the arsenate-containing medium were determined with an atomic absorption spectrophotometer. The intracellular levels of arsenic in the arsenate-resistant mutants were all lower than that of the parent strain CC425. Some of the arsenate-sensitive mutants, AS1 and AS3, showed obviously higher levels of arsenic than that of CC425, while other sensitive mutant, AS2, did not accumulate arsenic so much. Analysis of the chemical species of arsenic suggested that inorganic arsenic was converted to dimethylarsinic acid (DMAA) in CC425. However, DMAA was hardly detected in AS2. The mechanisms of the resistance to arsenate are discussed on its uptake and detoxification.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

Rediagnosis of the monotypic genus Lepadicyathus Prokofiev 2005 (Gobiesocidae: Diademichthyinae) and redescription of Lepadicyathus minor (Briggs 1955), new combination

Ichthyological Research - A revised diagnosis is provided for the poorly known clingfish genus Lepadicyathus Prokofiev 2005. The genus belongs to Diademichthyinae (sensu Conway et al.) and is...     

Rat tripeptidyl peptidase I: molecular cloning, functional expression, tissue localization and enzymatic characterization

We purified tripeptidyl peptidase I (TPP I) to homogeneity from a rat kidney lysosomal fraction and determined its physicochemical properties, including its molecular weight, substrate specificity and partial amino acid sequence. The molecular weight of the enzyme was calculated to be 280,000 and 290,000 by non-denaturing PAGE and gel filtration, respectively, and to be 43 000 and 46 000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of six identical subunits. The Km, Vmax, kcat and kcat/Km values of TPP I at optimal pH (pH 4.0) were 680 microM, 3.7 micromol x mg(-1) x min(-1), 33.1 s(-1) and 4.87 x 10(4) s(-1) x M(-1) for Ala-Ala-Phe-MCA, respectively. TPP I was significantly inhibited by PCMBS and HgCl2, and moderately by DFP. These findings also suggest that TPP I is an exotype serine peptidase that is regulated by SH reagent. TPP I released the tripeptide Arg-Val-Tyr from angiotensin III more rapidly than from Ala-Ala-Phe-MCA, and also released Gly-Asn-Leu from neuromedin B with the same velocity as from Ala-Ala-Phe-MCA. Angiotensin III and neuromedin B have recently been found to be good natural substrates for lysosomal TPP I. Furthermore, we determined the rat liver cDNA structure and deduced the amino acid sequence. The cDNA, designated as lambdaRTI-1, is composed of 2485 bp and encodes 563 amino acids in the coding region. By Northern blot analysis, the order for TPP I mRNA expression was kidney > or = liver > heart > brain > lung > spleen > skeletal muscle and testis. In parallel experiments, the TPP I antigen was detected in various rat tissues by immunohistochemical staining.     

The primary structure of human H-protein of the glycine cleavage system deduced by cDNA cloning

A full-length cDNA encoding the human H-protein of the glycine cleavage system has been isolated from a lambda gt11 human fetal liver cDNA library. The cDNA insert was 1091 base pairs with an open reading frame of 519 base pairs which encoded a 125-amino acid mature human H-protein with a 48-amino acid presequence. Human H-protein is 97%, 86%, and 46% identical to the bovine, chicken, and pea H-protein, respectively.     

Dynamics of hepatitis B virus quasispecies in association with nucleos(t)ide analogue treatment determined by ultra-deep sequencing

Background and Aims

Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity.

Methods

To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases.

Results

Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA.

Conclusion

Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.     

Adaptation changes in dynamic postural control and contingent negative variation during repeated transient forward translation in the elderly

Background

Adaptation changes in postural control and contingent negative variation (CNV) for the elderly were investigated during repeated forward floor translation.

Methods

Fifteen healthy elderly persons, living in the suburban area of Kanazawa City, Japan, underwent backward postural disturbance by a forward-floor translation (S2) 2 s after an auditory warning signal (S1). A set with 20 trials was repeated until a negative peak of late CNV was recognized in the 600-ms period before S2, and the last set was defined as the final set. Electroencephalograms, center of foot pressure in the anteroposterior direction (CoPap), and electromyograms of postural muscles were analyzed.

Results

CoPap displacement generated by the floor translation was significantly decreased until the twelfth trial in the first set, and mean CoPap displacement was smaller in the second and final sets than in the first set. The mean displacement was significantly smaller in the final set than the previous set. A late CNV with a negative peak was not recognized in the first and second sets. However, most subjects (13/15) showed a negative peak by the fourth set, when the late CNV started to increase negatively from about 1,000 ms after S1 and peaked at about 300 ms before S2. At about 160 ms before the CNV peak, the CoPap forward shift started. The increase in timing of the gastrocnemius activity related to the CoPap shift was significantly correlated with the CNV peak timing (r = 0.64). After S2, peak amplitudes of the anterior postural muscles were significantly decreased in the final set compared to the first set.

Conclusions

It was demonstrated that even for the elderly, with so many repetitions of postural disturbance, a late CNV with a negative peak was recognized, leading to accurate postural preparation. This suggests the improvement of frontal lobe function (e.g., anticipatory attention and motor preparation) in the elderly.     

Endogenous polyamines are intimately associated with highly condensed chromatin in vivo. A fluorescence cytochemical and immunocytochemical study of spermine and spermidine during the cell cycle and in reactivated nuclei

Polyamines are low molecular weight aliphatic polycations essential for cell proliferation and differentiation. By immunocytochemistry, as well as by two independent fluorescence cytochemical methods, we show that polyamines are associated with highly condensed chromatin in nucleated erythrocytes and in metaphase and anaphase chromosomes. In other cells, polyamines mainly occur in cytoplasm. The association between polyamines and DNA in condensed chromatin is so close that DNase treatment is necessary for making polyamines available for reaction with antibodies. Studies of chick/HeLa cell heterokaryons reveal that polyamines disappear from the chick erythrocyte nuclei concomitantly with DNA decondensation and initiation of RNA synthesis. Our data strongly suggest that polyamines are important for chromatin condensation in vivo.