|
1996 |
Sequestration of mitotic (M-phase) chromosomes in autophagosomes: mitotic programmed cell death in human Chang liver cells induced by an OH* burst from vanadyl(4). |
Sit, K. H.; Paramanantham,
R.; Bay, B. H.; Chan, H. L.; Wong,
K. P.; Thong, P.; Watt, F. |
Anat Rec, VOL. 245,
1996 May, PP. 1-8 |
BACKGROUND: Fragmentation
of genomic DNA in apoptosis/programmed
cell death (PCD) is a characteristic
hallmark in which both 2N and 4N DNA
from G1, S, and G2/M cell cycle phases
were seen degraded to the sub-2N Ao
level in PCD such as from serum deprivation,
glucocorticoid treatment, and gamma-radiation.
However M-phase (mitotic) cells are
said to perish only via non-programmed
or necrotic cell death unless they
were allowed to complete cytokinesis
and re-enter interphase. The morphological
criteria of PCD refer only to interphase
cells with intact nuclear membranes,
none seems applied to mitotic cells.
We show here autophagic sequestration
of mitotic chromosomes in a typical
PCD response where G1, S, and G2/M
DNA were replaced by a sub-2N Ao peak,
suggesting that mitotic cells may yet
have the option of PCD or suicide.
Autophagy is absent in necrosis. METHODS:
Mitotic human Chang liver cells in
normal monolayer culture were compared
with apoptotic counterparts initiated
by a burst of hydroxyl free radicals
(OH*) generated from vanadium internalized
by an NH4Cl prepulse containing vanadyl(4)
ions. Total (free and bound) vanadium
uptake was quantitated by elemental
spectral analysis of single cells using
a) Particle-Induced X-ray Emission
(PIXE) profiling, and b) Scanning Transmission
Ion Microscopy (STIM) in the nuclear
microscope. The Coulter EPICS PROFILE
II flow cytometer was used for a) the
cell cycle analysis using propidium
iodide-DNA binding, b) intracellular
pH (pHi) evaluation in the acidification-and-recovery
cycle, using ratiometric 2',7'-bis(2-carboxyethyl)-5
(and-6)-carboxyfluorescein (BCECF)
fluorescence quantitation. Transmission
electron microscopy examined the morphological
changes. Vanadyl(4)- generated hydroxyl
free radicals (OH*) were evaluated
by measuring OH*-benzoic adduct fluorescence
at 304/413 nm using the SPEX Fluoromax
photon counting spectrofluorometer.
RESULTS: Nuclear microscopy showed
that a 30 min acidification prepulse containing
4mM vanadyl(4) ions, V(4), had increased
the total (free and bound) vanadium
concentration of human Chang liver
cells from normal ultratrace levels
to 56,992 ppm of dry wt (1.1174 Eq
per kg dry wt). After the prepulse,
cells realkalinized in DMEM growth
medium, recovering to the physiological
pHi level in 30 min. At the physiological
pH 7 level, V(4) generated a burst
of OH* free radicals in the order of
15,000 folds above the prepulse (pH
4.5) level. In these conditions, spectrofluorometric
evaluation showed loss of DNA intercalation
with propidium iodide (PI-DNA binding)
indicating DNA degradation. Cell-by-cell
evaluation of the PI-DNA binding by
flow cytometry showed abolition of
G1, S, and G2/M phases and their replacement
by a sub-2N Ao peak of fragmented DNA,
emulating serum deprivation PCD in
these cells. Immediately upon initiating
an OH* burst ultrastructural profiles
showed mitotic chromosomes (M-phase
chromatin) being surrounded by rough
endoplasmic reticulum (RER) and small
vesicles, indicating their sequestration
in autophagosomes. Autophagy was also
seen to be a prominent feature in serum
deprivation PCD. CONCLUSION: Sequestration
of mitotic chromosomes by autophagosomes
in a typical PCD response showed a
well-defined morphological pathway
for direct degradation of M-phase chromatin
without first completing cytokinesis.
Mitotic cells could commit suicide
via autophagy directed at its own chromatin.
Autophagic sequestration of chromatin
in PCD is novel. |
|
1996 |
Selective fluorimetric recognition of dihydrogen phosphate over chloride anions by a novel ruthenium(II) bipyridyl receptor complex. |
Beer, P. D.; Mortimer,
R. J.; Szemes, F.; Weightman, J. S. |
Anal. Commun., VOL.
33, NO. 10, Oct 1996, PP. 365-366 |
The exclusive selectivity
to dihydrogen phosphate over chloride
ions by the novel mono(ligand) ruthenium
(II) complex ÝRuL(bipy)2¨ÝPF6¨2 where
L = Ý4,4'-bis(2-methoxyethyl)carbonylamino¨-2,2'-bipyridine
is studied. A HP 8452A diode-array
spectrophotometer and a Spex Fluoromax
instrument were used to record absorption
and emission spectra, respectively.
All measurements were made at 22.+-.2.degree.
C in deoxygenated acetonitrile solutions.
NMR spectra were obtained using a bruker
AM300 instrument with the solvent deuterium
signal as an internal reference. |
|
1995 |
Synchronous fluorescence spectroscopy of waste water and some potential constituents. |
Ahmad, S. R.; Reynolds, D. M. |
Water Res., VOL. 29,
NO. 6, Jun 1995, PP. 1599-1602 |
Conventional and synchronous
fluorescence spectroscopy were performed
with use of a FLUOROMAX SPEX instrument.
Excitation and emission monochromators
were mounted in a Czerny Turner configuration.
Synchronous fluorescence spectra were
recorded with use of an off-set wavelength
of 20 nm. A fluorescence peak at 340
nm and synchronous fluorescence peaks
at 280 nm were attributed to biodegradable
aromatic amino-acids from dissolved
organic matter and suspended solids
in the sewage. The major synchronous
fluorescence peak at 380 nm was attributed
to a non-biodegradable component. Results
are discussed. |
