Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Cholinesterases and cell proliferation in “nonstratified” and “stratified” cell aggregates from chicken retina and tectum

Summary Dissociated single cells from chicken retina or tectum kept in rotation-mediated cell culture aggregate, proliferate and establish a certain degree of histotypical cellto-cell relationships (sorting out), but these systems never form highly laminated aggregates (nonstratified R- and T-aggregates). In contrast, a mixture of retinal plus pigment epithelial cells forms highly stratified aggregates (RPE-aggregates, see Vollmer et al. 1984). The present comparative study of stratified and nonstratified aggregates enables us to investigate the process of cell proliferation uncoupled from that of tissue stratification. Here we try to relate these two basic neurogenetic processes with patterns of expression of cholinesterases (AChE, BChE) during formation of both types of aggregates.During early aggregate formation, in both stratified and nonstratified aggregates an increased butyrylcholinesterase activity is observed close to mitotically active cells. Quantitatively both phenomena show their maxima after 2–3 days in culture. In contrast, AChE-expression in all systems increases with incubation time. In nonproliferative areas, in the center of RPE-aggregates, the formation of plexiform layers is characterized initially by weak BChE and then strong AChE-activity. These areas correspond with the inner (IPL) and outer (OPL) plexiform layers of the retina in vivo. Although by sucrose gradient centrifugation we find that the 6S- and the fiber-associated 11S-molecules of AChE are present in all types of aggregates, during the culture period the ratio of 11S/6S-forms increases only in RPE-aggregates, which again indicates the advanced degree of differentiation within these aggregates.It is thus demonstrated that cholinesterases first correlate with neuronal cell proliferation and later with stratification, which indicates functions of both enzymes during both developmental periods.Abbreviations AChE acetylcholinesterase - BChE butyrylcholinesterase - iso-OMPA specific inhibitor of BChE - BW 284C51 specific inhibitor of AChE - IPL inner plexiform layer - OPL outer plexiform layer     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these photoreceptor proteins in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen and-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa

Cells from the slime variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER Endoplasmic reticulum - UDP-GlcNAc uridine-5-diphosphate N-acetyl glucosamine - GlcNAc N-acetyl glucosamine - SDS sodium dodecyl sulfate - PMSF phenyl methyl sulfonyl fluoride - EDTA ethylene diamino tetraacetic acid Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA hybridization techniques showed Chlorella fusca var. vacuolata and C. kessleri to be homogeneous species with DNA homologies of 90–100% C. fusca var. fusca and var. rubescens, however, have only about 15% DNA homology with C. fusca var. vacuolata and should no longer be regarded as varieties. A good correlation was found so far between biochemical and physiological characters used in the taxonomy of Chlorella and DNA relatedness. Mutant strains of Chlorella were tested for DNA homologies to prove the reliability of the taxonomical interpretation.     

Muscle damage and repair in voluntarily running mice: strain and muscle differences

Summary Soleus, extensor digitorum longus and tibialis anterior muscles of mice voluntarily running in wheels for periods of 5 to 120 days were studied in spaced serial and serial cross-sections. Shortly after the onset of running and during the next 2 weeks, degeneration, necrosis, phagocytosis and regeneration of muscle fibers, satellite cell proliferation and cellular infiltration were found in soleus muscles of mice from all strains investigated (CBA/J, NMRI, C57b, NIH, SWS and Balb/c). Tibialis anterior but not extensor digitorum longus muscles were also damaged. Predominantly high-oxidative fibers were affected (both slow-oxidative and fast oxidative glycolytic in soleus, fast-oxidative glycolytic in tibialis anterior). Denervated soleus muscles that had been passively stretched during running were not damaged. Evidence was found that, during the early period of running, split fibers form by myogenesis within (regeneration) or outside (satellite cell proliferation) necrotic muscle fiber segments. Split fibers persisted in solei of long-term (2 to 3 months) exercised CBA/J but not NMRI mice. In 6 out of 20 solei of CBA/J runners exercised for 2 months or longer, fiber-type grouping was observed in the areas where extensive damage usually occurred in the early periods. The results show that different muscles are damaged and repaired to varying degrees and that marked interstrain and inter-individual differences are present. It appears that acute muscle injury occurring upon onset of voluntary running is a usual event in the adaptation of muscles to altered use.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Acquisition of a brief behavioral experience in the presence of neuron-specific and D2-CAM/N-CAM-specific antisera

The effect of intraventricular infusion of D2-CAM/N-CAM directed antibodies prior to the acquisition of a passive-avoidance paradigm is described. The antisera used in this study were the neuron specific anti-BPM and a D2-CAM/N-CAM specific serum, anti-D2. Anti-BPM reliably inhibited paradigm acquisition when recall was ascertained at 24 and 48 hours and no effect was noted with absorbed anti-BPM or in sham-operated animals. This effect was time-dependent and no inhibition of memory formation was noted when the antiserum was administered at 6 and 10 hours after training. In contrast, infusion of anti-D2 had no effect on paradigm acquisition. These findings are discussed in relation to the potential synaptogenic events associated with memory formation.