Isolabelling and Chromosome Strandedness

IF cells of eukaryotes are pulse-labelled with tritiated thymidine and then allowed to pass through mitosis (M₁) and complete a second round of DNA replication (S₂) without label, at the next mitosis (M₂) the label is seen to segregate semi-conservatively; that is, with the exception of sister chromatid exchanges, all the label will pass to one chromatid^1–4.     

l-glutamine is a key parameter in the immunosuppression phenomenon

The present work describes the influence of both vitamin C (VC) and mechanical stimulation on development of the extracellular matrix (ECM) and improvement in mechanical properties of a chondrocyte-agarose construct in a regenerating tissue disease model of hyaline cartilage. We used primary bovine chondrocytes and two types of VC, ascorbic acid (AsA) as an acidic form and ascorbic acid 2-phosphate (A2P) as a non-acidic form, and applied uniaxial compressive strain to the tissue model using a purpose-built bioreactor. When added to the medium in free-swelling culture conditions, A2P downregulated development of ECM and suppressed improvement of the tangent modulus more than AsA. By contrast, application of mechanical stimulation to the construct both increased the tangent modulus more than the free-swelling group containing A2P and enhanced the ECM network of inner tissue to levels nearly as high as the free-swelling group containing AsA. Thus, mechanical stimulation and strain appears to enhance the supply of nutrients and improve the synthesis of ECM via mechanotransduction pathways of chondrocytes. Therefore, we suggest that mechanical stimulation is necessary for homogenous development of ECM in a cell-associated construct with a view to implantation of a large-sized articular cartilage defect.     

ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon

We have measured the transbilayer diffusion of spin-labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in pig lymphocyte plasma membrane. At 4 degrees C and 37 degrees C the aminophospholipids are rapidly transported from the outer to the inner leaflet of the membrane, whereas the choline-containing phospholipids experience a slower diffusion. This selectivity is abolished after cell treatment by SH-group reagents indicating that the aminophospholipid translocation is protein-dependent and must be driven by a system analogous to the one existing in the human red cell membrane. The fact that the selectivity exists at low temperature, that it does not depend on cytoskeleton integrity and that there is a competition between the two aminophospholipids show that this translocation is not purely an endocytic process.     

Convergent evolution of enzyme active sites is not a rare phenomenon

Since convergent evolution of enzyme active sites was first identified in serine proteases, other individual instances of this phenomenon have been documented. However, a systematic analysis assessing the frequency of this phenomenon across enzyme space is still lacking. This work uses the Query3d structural comparison algorithm to integrate for the first time detailed knowledge about catalytic residues, available through the Catalytic Site Atlas (CSA), with the evolutionary information provided by the Structural Classification of Proteins (SCOP) database. This study considers two modes of convergent evolution: (i) mechanistic analogues which are enzymes that use the same mechanism to perform related, but possibly different, reactions (considered here as sharing the first three digits of the EC number); and (ii) transformational analogues which catalyse exactly the same reaction (identical EC numbers), but may use different mechanisms. Mechanistic analogues were identified in 15% (26 out of 169) of the three-digit EC groups considered, showing that this phenomenon is not rare. Furthermore 11 of these groups also contain transformational analogues. The catalytic triad is the most widespread active site; the results of the structural comparison show that this mechanism, or variations thereof, is present in 23 superfamilies. Transformational analogues were identified for 45 of the 951 four-digit EC numbers present within the CSA and about half of these were also mechanistic analogues exhibiting convergence of their active sites. This analysis has also been extended to the whole Protein Data Bank to provide a complete and manually curated list of the all the transformational analogues whose structure is classified in SCOP. The results of this work show that the phenomenon of convergent evolution is not rare, especially when considering large enzymatic families.     

Hypoxic preconditioning is a phenomenon increasing tolerance of cardiomyocytes to hypoxia-reoxygenation

The work covers the problem of hypoxic preconditioning (HP) carried out in isolated cardiomyocytes. Papers on delayed HP in vivo are comparatively few, and only some single works are devoted to early preconditioning in vivo. It has been established that the HP limits necrosis and apoptosis of cardiomyocytes and improves contractility of the isolated heart after ischemia (hypoxia) and reperfusion (reoxygenation). It was found that adenosine was a trigger of iP in vitro. It was proved that NO* was a trigger of HP both in vitro and in vivo. It was shown that reactive oxygen species also were triggers of hypoxic preconditioning. It was shown that ERK1/2 and p38 kinase played important role in delayed HP in vitro.     

Mitochondrial targeting of farnesyl diphosphate synthase is a widespread phenomenon in eukaryotes

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat(2)) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat(2) showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat(2). Cellular internalization of Tat and Tat(2) was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 degrees C) remarkably increased cellular internalization of Tat as well as Tat(2) but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 muM), chloroquine (100 muM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat(2). These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat(2) suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.     

Heat shock-induced apoptosis in preimplantation bovine embryos is a developmentally regulated phenomenon

Apoptosis is a form of cell death that can function to eliminate cells damaged by environmental stress. One stress that can compromise embryonic development is elevated temperature (i.e., heat shock). For the current studies, we hypothesized that heat shock induces apoptosis in bovine embryos in a developmentally regulated manner. Studies were performed to 1) determine whether heat shock can induce apoptosis in preimplantation embryos, 2) test whether heat-induced apoptosis is developmentally regulated, 3) evaluate whether heat shock-induced changes in caspase activity parallel patterns of apoptosis, and 4) ascertain whether exposure to a mild heat shock can protect embryos from heat-induced apoptosis. As determined by TUNEL reaction, exposure of bovine embryos > or =16 cells on Day 5 after insemination to 41 or 42 degrees C for 9 h increased the percentage of cells undergoing apoptosis. In addition, there was a duration-dependent increase in the proportion of blastomeres that were apoptotic when embryos were exposed to temperatures of 40 or 41 degrees C, which are more characteristic of temperatures experienced by heat-stressed cows. Heat shock also increased caspase activity in Day 5 embryos. However, heat shock did not induce apoptosis in 2- or 4-cell embryos, nor did it increase caspase activity in 2-cell embryos. The apoptotic response of 8- to 16-cell-stage bovine embryos to heat shock depended upon the day after insemination that heat shock occurred. When 8- to 16-cell embryos were collected on Day 3 after insemination, heat shock of 41 degrees C for 9 h did not induce apoptosis. In contrast, when 8- to 16-cell embryos were collected on Day 4 after insemination and exposed to heat shock, there was an increase in the percentage of cells undergoing apoptosis. Exposure of 8- to 16-cell embryos at Day 4 to a mild heat shock of 40 degrees C for 80 min blocked the apoptotic response to a subsequent, more-severe heat shock of 41 degrees C for 9 h. In conclusion, apoptosis is a developmentally acquired phenomenon that occurs in embryos exposed to elevated temperature, and it can be prevented by induced thermotolerance.     

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.     

Incubation of conditional suppression is an associatively-based phenomenon

Ten rats were deprived of water and trained to lick a tube for saccharin reinforcement. In each of the two sessions that followed, the rats received six contiguous pairings of a 30-second illumination of the houselight and a 0.75 second, 0.10 mA electric shock while licking. No sign of conditioning was observed during the first experimental session, but profound conditioning was observed on the first and subsequent trials of the second conditioning session. No comparable change in the rate of licking was observed in groups of rats that received only presentations of the visual stimulus, only presentations of the electric shock, or random presentation of the visual stimulus and electric shock during the first conditioning session. These data establish that the incubation of conditional suppression is an associative phenomenon.     

Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Urinary retention and the lunisolar cycle: is it a lunatic phenomenon?

OBJECTIVE--To determine whether a relation between urinary retention and temporal rhythms exists. DESIGN--Retrospective analysis of patients presenting over three years. SETTING--Urology departments in two hospitals. PATIENTS--815 Patients presenting as emergency admissions with urinary retention and requiring immediate decompression of the bladder. MAIN OUTCOME MEASURES--Calendar date of each admission to determine circadian, monthly, and seasonal periodicity. RESULTS--No association was found between urinary retention and circadian, monthly, or seasonal rhythms. A significantly higher (p less than 0.001) incidence of urinary retention was observed during the new moon in comparison with other phases of the lunar cycle. CONCLUSIONS--Urinary retention is periodic in nature. This should be considered when the workload of a specialist urological department is organised.     

Desquamation is a novel phenomenon for collective prostate epithelial cell deletion after castration

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell–cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.     

Nonuniform behavior of multiple isoactins in the same cell is a cell-dependent phenomenon

The functional significance of multiple isoactins in the same cell is still not understood. To address this question, we examined the response of smooth muscle and cardiac muscle alpha-isoactins to a serial extraction procedure applied to both muscle and nonmuscle cell types. We compared these extraction results with results obtained with the beta- and gamma-nonmuscle actin isoforms from the same cells. In differentiated BC3H1 nonfusing muscle cells (smooth muscle alpha-isoactin), in human rhabdomyosarcoma cells (cardiac alpha-isoactin), and in chick skeletal muscle cells (cardiac alpha-isoactin), different fractions were found selectively enriched in either the nonmuscle or the muscle-specific actin isoforms compared with their relative abundance in whole cell extracts. Conversely, when these same isoactins were examined either in undifferentiated BC3H1 cells or in mouse nonmuscle cells stably transfected with a cardiac alpha-isoactin gene, no enrichment of these isoforms above their relative abundance in whole cell extracts was observed. These results indicate that within the muscle or muscle-like cells examined, the different actin isoforms were either selectively utilized or localized. These results further show that isoactin-specific responses observed were apparently related to the cell type in which they were found and not to differences in inherent physical properties such as solubility of the different isoactins examined.     

Heterogenous stomatal closure in response to leaf water deficits is not a universal phenomenon

The extent and occurrence of water stress-induced “patchy” CO₂ uptake across the surface of leaves was evaluated in a number of plant species. Leaves, while still attached to a plant, were illuminated and exposed to air containing [¹⁴C]CO₂ before autoradiographs were developed. Plant water deficits that caused leaf water potential depression to −1.1 megapascals during a 4-day period did result in heterogenous CO₂ assimilation patterns in bean (Phaseolus vulgaris). However, when the same level of stress was imposed more gradually (during 17 days), no patchy stomatal closure was evident. The patchy CO₂ assimilation pattern that occurs when bean plants are subjected to a rapidly imposed stress could induce artifacts in gas exchange studies such that an effect of stress on chloroplast metabolism is incorrectly deduced. This problem was characterized by examining the relationship between photosynthesis and internal [CO₂] in stressed bean leaves. When extent of heterogenous CO₂ uptake was estimated and accounted for, there appeared to be little difference in this relationship between control and stressed leaves. Subjecting spinach (Spinacea oleracea) plants to stress (leaf water potential depression to −1.5 megapascals) did not appear to cause patchy stomatal closure. Wheat (Triticum aestivum) plants also showed homogenous CO₂ assimilation patterns when stressed to a leaf water potential of −2.6 megapascals. It was concluded that water stress-induced patchy stomatal closure can occur to an extent that could influence the analysis of gas exchange studies. However, this phenomenon was not found to be a general response. Not all stress regimens will induce patchiness; nor will all plant species demonstrate this response to water deficits.     

No gene is an island: the flip-flop phenomenon

An increasing number of publications are replicating a previously reported disease-marker association but with the risk allele reversed from the previous report. Do such "flip-flop" associations confirm or refute the previous association findings? We hypothesized that these associations may indeed be confirmations but that multilocus effects and variation in interlocus correlations contribute to this flip-flop phenomenon. We used theoretical modeling to demonstrate that flip-flop associations can occur when the investigated variant is correlated, through interactive effects or linkage disequilibrium, with a causal variant at another locus, and we show how these findings could explain previous reports of flip-flop associations.     

Histone H1 dephosphorylation is mediated through a radiation-induced signal transduction pathway dependent on ATM.

Ionizing radiation is known to activate multiple signal transduction pathways, but the targets of these pathways are poorly understood. Phosphorylation of histone H1 is thought to have a role in chromatin condensation/decondensation, and we asked whether ionizing radiation (IR) would alter H1 phosphorylation. Our data demonstrate that low doses of IR result in a dramatic, but transient, dephosphorylation of H1 isoforms. The in vivo IR-induced dephosphorylation of H1 is completely blocked by wortmannin and is abrogated in ataxia telangiectasia cells. Furthermore, we measured radiation-induced inhibition of cyclin dependent kinase activity and activation of histone H1 phosphatase activity. Both activities were affected by radiation-induced signals in an ATM-dependent manner. Thus, the rapid IR-induced dephosphorylation of H1 involves a pathway including ATM and a wortmannin-sensitive step leading to both inhibition of cyclin-dependent kinase activities as well as activation of H1 phosphatase(s).