Organization of centromeres in the decondensed nuclei of mature human sperm

The localization of centromeres in mature human sperm was shown by immunofluorescent labeling and nonisotopic in situ hybridization. In the decondensed nucleus structural elements (dimers, tetramers, linear arrays and V shape structures) formed by individual centromeres of nonhomologous chromosomes were observed. They organize the compact chromocenter, which was shown for nuclei decondensed to a low extent. The chromocenter is buried inside the nucleus; in contrast, telomeric regions of chromosomes were tentatively localized on the periphery. Thus, a gross architecture, which can influence selective unpackaging of the paternal genome upon fertilization, exists in human sperm.     

Microwave influence on the isolated heart function: II. Combined effect of radiation and some drugs

The combined effects of microwave radiation and some drugs were studied in an isolated frog auricle preparation. The experiments established that exposure to pulse-modulated 915 MHz microwaves for up to 40 min had no effect on either the rate or the amplitude of spontaneous auricle twitches, unless the average absorbed power was high enough to produce preparation heating. Treatment of the preparation with saline containing (0.6–3.0) 10^?5 M of propranolol or (0.5–1.5) 10^?7 M of atropine altered neither its pacemaker nor its contractile functions; these drugs also had no effect when they were combined with nonthermal microwave irradiation. Caffeine (1 mM) strongly increased the average heart power, which was calculated as the product of twitch rate and amplitude. The caffeine effect appeared to be significantly augmented (by about 15%, P<0.02) under exposure to burst-type pulsed microwaves (pulse width, 1.5 msec; pause, 2.5 msec; 8 pulses/burst, 16 bursts/s; average SAR, 8–10 W/kg). By itself, this modulation was not effective; the heating of the preparation and saline during exposure was approximately 0.1°C, which could not account for the detected changes. The experimental results demonstrate that caffeine treatment increases the microwave sensitivity of the frog auricle preparation and reveals primarily subthreshold, nonthermal microwave effect. © 1995 Wiley-Liss, Inc.     

HSP70 Translocates into a cytoplasmic aggregate during lymphocyte activation

The percentage of T and B lymphocytes expressing a distinct cytoplasmic aggregate enriched in spectrin, ankyrin, and in several other proteins including protein kinase C greatly increases following various activation protocols. Members of the 70 kDa family of heat shock proteins (hsp70) temporarily bind to and stabilize unfolded segments of other proteins, a function apparently required for proper protein folding and assembly. Considering the multiprotein and dynamic nature of the lymphocyte aggregate, the possibility that hsp70 also might be associated with componets of this structure is considered here. Double immunofluorescence analysis indicates that hsp70 is a component of the lymphocyte aggregate and is coincident with spectrin in a subpopulation of freshly isolated, untreated lymphocytes from various murine tissues and in a T-lymphocyte hybridoma. When cell lysates of lymph node T cells are immunoprecipitated using an antibody against hsp70 or spectrin and then analyzed by Western blot utilizing the alternate antibody, it was found that hsp70 and spectrin coprecipitated with one another. Moreover, this coprecipitation could be abolished by addition of ATP. This latter observation was extended to lymphoid cells using a transient permeabilization procedure, and it was shown that addition of exogenous ATP results in the dissipation of the aggregate structure itself. Finally, conditions that result in T-cell activation and aggregate formation, i.e., treatment with the phorbol ester PMA or T-cell receptor cross-linking, also lead to the repositioning of hsp70 into the aggregate from a membrane/cytosolic locale in congruence with spectrin. These data suggest that hsp70 is an active component of the aggregate and that it may function in the interactions believed to occur in this unique activation-associated organelle. © 1995 Wiley-Liss, Inc.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.     

Non‐canonical roles of tRNAs and tRNA mimics in bacterial cell biology

Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl‐tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl‐tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non‐canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.     

Chromosome demise in the wake of ligase-deficient replication

Bacterial DNA ligases, NAD⁺‐dependent enzymes, are distinct from eukaryotic ATP‐dependent ligases, representing promising targets for broad‐spectrum antimicrobials. Yet, the chromosomal consequences of ligase‐deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase‐deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double‐strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non‐allelic double‐strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double‐strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase‐deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double‐strand breaks and then into irreparable double‐strand gaps may be behind lethality of any DNA damaging treatment.