Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.     

Evaluation of the antibacterial activity in vitro of various cephalosporins on different gram-negative bacterial species of recent clinical isolation

The Authors have compared the antibacterial activity "in vitro" of new cephalosporines (cephuroxim, cephoxitin and cephaclor) with other cephalosporines having an action which is already known (cephaloridine, cephazoline and cephalexine) on the Gram-negative bacterial strains of recent clinical isolation. The results show that the cephalosporines of last generation, having in their molecular nucleus a methoximinic group, realize a greater antibacterial protection than those cephalosporines of antecedent preparation.     

A stereochemical model for phospholipids. II: Conformational analysis and molecular packing of phosphatidylethanolamine (PE)

A partition energy method procedure was applied to select the energetically favoured conformations of phosphatidylethanolamine (PE) as polar constituents of phospholipid molecules. The result indicated a large degree of freedom for the two torsion angles of the ester bond of the phosphate and a gauche, gauche star conformation for the ethane bond.A packing process of the molecule was carried out through a potential energy calculation by considering the conformers selected above, using previously published procedure and conventions. All the arrangements which possess the best packing energy values were characterised by an orientation of the PN dipolar segment parallel to the lattice plain. Rotation of the internal torsion angles and rotation in the eulerian space of the molecule produced differences in the charged groups that interact. An additional minimum was present in the energy packing process of those conformers which have the first torsion angle of the phosphate in a trans conformation. This minimum, which corresponds to an orientation of the molecule orthogonal to the lattice plane, requires a complete neutralisation of the point charges on the system.The results of the calculation underline the importance of changes in the behaviour of the polar group of the phospholipids in the packing process.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Optimization of accessible catalase activity in polyacrylamide gel-immobilized Saccharomyces cerevisiae

The permeabilization of Saccharomyces cerevisiae (baker's yeast), either before or after immobilization in polyacrylamide gel (PAG), has been examined as a means to increase the catalase activity of PAG-immobilized yeast cells. Prior permeabilization of the cells resulted in large losses of catalase activity during immobilization, but permeabilization after immobilization produced increases in the catalase activity of yeast/PAG particles. A dependence of the accessible catalase activity on the concentration of polyacrylamide in permeabilized yeast/PAG particles, and on the method of permeabilization of the immobilized cells, was observed. Optimal levels of stable catalase activity (1000-2000 IU/g PAG particles; ca. 5%-10% of total available activity) were obtained by immobilizing yeast cells (0.5 g wet cells/mL gel) in 10% (w/v) PAG, followed by permeabilization of the entrapped cells with either cetyltrimethylammonium bromide, Triton X-100 and one freeze-thaw, or five freeze-thaw cycles. (c) 1992 John Wiley & Sons, Inc.     

Diurnal changes in psychophysiological variables of school girls: comparison with regard to age and teacher's appreciation of learning.

Ninety-five nonresident girls of a private school volunteered for the study with the teachers' help as well as parental consent. Ages were approximately 8, 9, and 10 years. They were synchronized with diurnal activity from 0730 to 2100 h and nocturnal rest. Fatigue, drowsiness, and attention were self-rated using visual analogue scales; oral temperature was self-measured and a letter cancellation test was performed. Each of these variables was measured at school at 0900, 1100, 1400, and 1600 h on Mondays, Thursdays, Fridays, and Saturdays for two consecutive weeks in 1987 (March 30-April 11) and again in 1989 (March 13-25) when the youngest group had become 10 years old. According to conventional teacher evaluation of learning (learning performance) within each group, three subgroups were formed: top third, middle third, and bottom third. Time series (more than 50,000 data) were analyzed according to several statistical methods, but mainly chronograms with ANOVA. Similar diurnal changes in oral temperature were validated for each group and subgroups. The occurrence of a diurnal change in self-rated variables (fatigue and drowsiness) and score in letter cancellation was age related: no detection in the 8-year-old group (and subgroups) and validation (p less than 0.002) in 9- and 10-year-old groups (and respective subgroups). A good learning performance was associated with a reduced drowsiness in school girls of 9 and 10 years. Age-related, time-of-day differences in drowsiness (when detected) as well as learning performance effect were not associated with observed duration of sleep. Validated changes in self-rated fatigue were close to that of drowsiness. At 0900 h, girls of 9 and 10 years were more tired when belonging to the bottom third than top third subgroup. Whatever the time of day, self-rated attention was greater in the top than in the bottom third for these girls. Differences related to learning performance were validated in each grade. However, best scores were recorded for the bottom third in the 8-year-old group, while best scores were provided by top third subgroups in 10-year-old girls. It seems that in girls around 8 years of age, critical changes can be detected with regard to the (ontogenic?) occurrence of time-of-day differences in a set of psychophysiologic variables as well as influential effects of learning performance on the same variables. Reported finding are compatible with the hypothesis of circadian oscillators working at the level of the cortex of the human brain.     

Physical and chemical scavenging of singlet molecular oxygen by tocopherols

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.     

Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)