Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.     

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads,as Determined by Sequence-Based Typing

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires'' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.     

Piperine analogs in a hydrophobic fraction from Piper ribersoides (Piperaceae) and its insect antifeedant activity

Piper ribersoides Wall. (Piperaceae), which is called “Khua Sa khan” in the local language, is mainly grown in Laos. This plant is used as a food in Laos, but no report on its metabolites exists. Crushed stems were immersed in methanol. The ethyl acetate fraction showed potential insect antifeedant activity for Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), and some of the active compounds were piperine analogs. Piperine and its geometrical isomer independently showed potent antifeedant activities, and piperine was presumably the main active compound in the methanol extract. Interestingly, we discovered that the antifeedant activity was reduced when they were mixed (50:50).     

Continuous ATP Regeneration Using Immobilized Yeast Cells

Arthrobacter simplex was screened as an α-keto-δ-guanidinovalerate (ketoarginine) assimilating organism. A characteristic feature was its growth on ketoarginine as a carbon source; it began to grow after an extremely long lag. Its growth was stimulated by addition of 0.02% yeast extract to the medium.

The results indicated the transamination of arginine-α-ketoglutarate (α-KGA) and the hydrolyzing reaction of ketoarginine into α-keto-δ-aminovalerate and urea. Two intermediates, ketoarginine and α-keto-δ-aminovalerate, were isolated and identified by various procedures. Coupling of the two reactions was demonstrated in cell-free extracts of arginine-grown cells; ketoarginine formed from arginine by transamination with α-KGA was hydrolyzed directly to α-keto-δ-aminovalerate and urea. The metabolic routes of arginine in microorganisms were discussed.     

DOPA Production with Enterobacter cloacae NB 320 by Transamination Reaction

Taxonomical investigation was performed on the bacterium, strain NB 320 isolated from soil, and it was identified as Enterobacter cloacae. This bacterium produced the enzyme which catalyzed the transamination reaction between 3,4-dihydroxyphenyl pyruvate and an amino acid to form l-Dopa.

The optimum culture conditions for the enzyme production were studied along with the characteristics of the enzyme. The enzyme of the strain was different in some properties from that of Alcaligenes faecalis IAM 1015 which had been already studied. The former utilized glutamate as an amino donor best among the amino acids tested for transamination and was induced by the addition of glutamine and asparagine. Intact cells of the strain did not catalyze the reaction unless they were treated with sonication or with a detergent.     

Studies on DOPA Transaminase of Alcaligenes faecalis

Some enzymatic properties were examined on the transaminase (DOPA transaminase) which catalyzes the reaction between 3,4-dihydroxy phenyl pyruvate (DOPP) and certain amino acids to form 3,4-dihydroxyphenyl-L-alanine (DOPA). The cell-free extract from Alcaligenes faecalis IAM 1015 was used as the DOPA transaminase. L-Aspartate, L-glutamate, and L-phenylalanine were utilized efficiently as amino donor. The occurrence of three kinds of transaminase—aspartate-DOPP transaminase (ADT), glutamate-DOPP transaminase (GDT), and phenylalanine-DOPP transaminase (PDT)—was postulated.

The pH optima of these enzymes were observed in the alkaline pH range. The enzymes were unstable in the acidic range and inactivated above 60°C. Ca²⁺, Mg²⁺, and Mn²⁺ protected PDT from heat denaturation. Fe²⁺, Cu²⁺, and Al³⁺ remarkably inhibited the enzyme reaction.     

Effects of sand burial depth on the root system of Salix cheilophila seedlings in Mu Us Sandy Land, Inner Mongolia, China

Salix cheilophila Schneid. is a naturally occurring Salix species in Mu Us Sandy Land, Inner Mongolia, China. We focused on the morphological adaptability of S. cheilophila to sand dune burial. For morphological measurements, 32 S. cheilophila seedlings were removed from a community which was in the process of being buried by a shifting sand dune. Each seedling collected included the entire root system. We measured the number, length, and biomass of the adventitious roots, primary lateral roots, and taproot, and compared the morphological characteristics of the root system, including adventitious roots, for seedlings buried to various levels in the sand. The growth range of adventitious roots increased as the length of the buried portion of the main shoot increased. In addition, the total dry weight of all current-year shoots tended to increase gradually with increasing total dry weight of the adventitious roots. These results suggest that S. cheilophila tends to make use of the sedimentary sand layer that accompanies shifting sand dunes. However, there was no correlation between biomass or number of adventitious roots and the length of the buried part of the main shoot. Thus, S. cheilophila does not grow adventitious roots proportional to the buried part. These morphological characteristics of the root system, including the adventitious roots, may indicate that S. cheilophila has poor morphological adaptability to sand dune burial.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.