Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Activities of three lysosomal enzymes--acid RNase. N-acetyl-beta-D-glucosaminidase and acid phosphatase--were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to fivefold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown also increased as cultures approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH4Cl, but the rate of turnover in the presence of NH4Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops.     

Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).     

Chloroplast division: squeezing the photosynthetic captive

Chloroplasts have evolved from a cyanobacterial endosymbiont and have been retained in eukaryotic cells for more than one billion years via chloroplast division and inheritance by daughter cells during cell division. Recent studies revealed that chloroplast division is performed by a large protein complex at the division site, encompassing both the inside and the outside of the two envelope membranes. The division complex has retained a few components of the cyanobacterial division complex to go along with other components supplied by the host cell. On the basis of the information about the division complex, we are beginning to understand how the division complex evolved, and how eukaryotic host cells regulate chloroplast division during proliferation and differentiation.     

Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells

Telomestatin is a potent G-quadruplex ligand that interacts with the 3' telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the gamma-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.     

Comments on ‘Is Partial Coherence a Viable Technique for Identifying Generators of Neural Oscillations?’

To aid prospective neural connectivity inference analysts and hoping to preclude misconception spread, we exploit the didatic value of some of the issues raised by Albo et al. (Biol Cybern 90: 318–326, 2004) who claim that signal-to-noise ratio (SNR) values can lead to mistakes in structural inference when using partial coherence in connection to Gersch’s 1970 method for spotting signal sources (Gersch in Math Biosci 14: 177– 196, 1972). We show theoretically that Gersch’s method is able only to spot which measurement of some common underlying factor has the least amount of additive noise and that this has nothing to do with any reasonable notion of ‘causality’ as suggested by Albo et al. (Biol Cybern 90: 318–326, 2004). We also show that despite the inherent structural ambiguity of the model used by Albo et al. (Biol Cybern 90: 318–326, 2004) to back their claim, its data can nonetheless furnish the correct time precedence hierarchy between the activities in its measured structures, both when simple (correlation) and more sophisticated methods are used (partial directed coherence) (Baccala and Sameshima in Biol Cybern 84:463–474, 2001a) in a true depiction of time series causality.     

Specific and sensitive enzymatic measurement of sphingomyelin in cultured cells

Sphingomyelin (SM) is the most abundant sphingolipid in mammalian cell membranes and plays multiple physiological roles. In this study, we improved the sensitivity of the enzymatic measurement of SM and validated its specificity and accuracy. The enzymatic reaction sequence of the method involves the hydrolysis of SM by sphingomyelinase, dephosphorylation of phosphorylcholine, oxidation of choline, and reaction of hydrogen peroxide with Amplex Red. The calibration curve was shown to be quadratic and linear at low (0-10μM) and high (10-100μM) concentrations, respectively, and the detection limit was 0.5μM (5pmol in the reaction mixture), which was more sensitive than all other SM assays reported previously. This SM measurement using Triton X-100 detected only SM, but not other choline-containing phospholipids, sphingosylphosphocholine, phosphatidylcholine, and lysophosphatidylcholine, and quantified SM regardless of the length and double bonds of the acyl chain. By using this method, we demonstrated that an increase in the density of HEK293 cells was accompanied by an elevation in the cellular content of SM, and that the treatment of HEK293 cells with tumor necrosis factor α significantly decreased the SM content. This specific and sensitive method for measuring SM will be helpful in studying various cellular processes.     

Effect of green tea powder (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L. cv. Benifuuki) particle size on <Emphasis Type="Italic">O</Emphasis>-methylated EGCG absorption in rats; The Kakegawa Study

Tea polyphenols, e.g., (-)-epigallocatechin-3-O-(3-O-methyl gallate (EGCG3”Me), (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-O-gallate (ECG), and (-)-epicatechin (EC), are believed to be responsible for the beneficial effects of tea. ‘Benifuuki’, a tea (Camellia sinensis L.) cultivar grown in Japan, is rich in the anti-allergic molecule epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3”Me). Pulverized Benifuuki green tea powder (BGP) is more widely distributed than leaf tea in Japan. Japanese people mix their pulverized tea with water directly, whereas it is common to drink leaf tea after extraction. However, few studies of the effects of BGP particle size on polyphenol bioavailability have been performed. This study was conducted to investigate the absorption of catechins in rats after the intragastric administration of Benifuuki green tea. Therefore, we assessed the plasma concentrations of catechins following the ingestion of BGP with different mean particle sizes (2.86, 18.6, and 76.1 μm) or Benifuuki green tea infusion (BGI) as a control in rats. The bioavailabilities of EGCG3”Me, EGCG, ECG, EGC, and EC were analyzed after the oral administration of a single dose of Benifuuki green tea (125 mg/rat) to rats. The plasma concentrations of tea catechins were determined by HPLC analysis combined with of electrochemical detection (ECD) using a coulometric array. The AUC (area under the drug concentration versus time curve; min μg/mL) of ester-type catechins (EGCG3”Me, EGCG, and ECG) for the BGP 2.86 μm were significantly higher than those in the infusion and 18.6 and 76.1 μm BGP groups, but the AUC of free-type catechins (EGC and EC) showed no differences between these groups. Regarding the peak plasma level of EGCG3”Me adjusted for intake, BGP 2.86 μm and BGI showed higher values than the BGP 18.6 and 76.1 μm groups, and the peak plasma levels of the other catechins displayed the same tendency. The present study demonstrates that the bioavailability of ester-type catechins (EGCG and ECG) can be improved by reducing the particle size of green tea, but the plasma level of EGCG3”Me in the BGI group was similar to that in the BGP 2.86 μm group. This result suggests that drinking Benifuuki green tea with a particle size of around 2 μm would deliver the anti-allergic EGCG3”Me and the anti-oxidant EGCG efficiently.     

Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Fetal breathing, sleep state, and cardiovascular responses to graded anemia in sheep

Graded anemia was produced for 2 h in 10 unanesthetized fetal sheep by infusing plasma in exchange for fetal blood. This reduced the mean fetal hematocrits during the 1st h of anemia to 19.7 +/- 0.5% [control (C) = 28.2 +/- 1.1%] for mild anemia, 17.4 +/- 0.9% (C = 30.0 +/- 1.1%) for moderate anemia, and 15.1 +/- 1.0% (C = 29.2 +/- 1.3%) for severe anemia. The respective mean arterial O2 contents (CaO2) were 4.46 +/- 0.20, 3.89 +/- 0.24, and 3.22 +/- 0.19 ml/dl. Mean arterial PO2 was reduced significantly (by 2 Torr) only during moderate anemia, and mean arterial pH was decreased only during severe anemia. No significant changes occurred in arterial PCO2. Fetal tachycardia occurred during anemia. Mean arterial pressure was reduced by 2-3 mmHg during mild anemia; however, no significant blood pressure changes were observed for moderate or severe anemia. The incidence of rapid-eye movements and breathing activity was not affected by mild anemia, but the incidence of both was reduced significantly during moderate and severe anemia. It is concluded that 1) a reduction in CaO2 of greater than 2.48 +/- 0.22 ml/dl by hemodilution inhibits rapid-eye movements and breathing activity, and 2) the PO2 signal for inhibition does not come from arterial blood but from lower PO2 in tissue.