Aristotel Pappelis

Nuclear area changes in senescing human diploid fibroblasts

The pattern of mean nuclear area changes was determined in human fibroblast cell strains from male donors of three different ages (8,40 and 84 years) during their in vitro lifespan. There was a statistically significant increase in mean nuclear area of cells of all three strains during their aging in vitro. A gradual increase of the subpopulation of cells with larger nuclei was also observed. Evidence for a positive correlation between aging in vitro, as reflected by mean nuclear areas at the tenth doubling from the end of in vitro doubling activity, and donor age is presented.

Effect of abscisic acid, gibberellic acid, indoleacetic acid, and kinetin on selective ribosomal cistron regulation in quiescent and senescent onion leaf base tissue

Small pieces of tissue from the basal, equatorial, near-apical, and apical regions of the third turgid onion leaf base were treated (3 and 6 h in the dark) with abscisic acid (ABA), gibberellic acid (GA3), indoleacetic acid (IAA), and kinetin (K) and compared with responses in water controls. ABA inhibited the activation (increase in size and changes in morphologies from round or oval to elongated-oval and dumbbell) of major nucleolar organizer regions (NORs) in basal, equatorial, and near-apical tissue. GA3 and K activated the major NORs in the basal, equatorial, and near-apical tissue. IAA stimulated the activation of major NORs in basal tissue but inhibited their activation in equatorial and near-apical tissue. No major nucleoli were activated in control or plant growth regulator-treated apical tissue. Minor NORs were not expressed in the control and plant growth regulator-treated tissue in these four locations. Actinomycin D and cycloheximide inhibited major NOR activation in equatorial control and kinetin-treated tissue. We propose that ABA, GA3, IAA, and K are major NOR regulators. We infer that the basal through near-apical cells were quiescent during post-harvest storage and that the cells in the apical tissue had senesced beyond the point of no return (degeneration of the karyoskeleton) in the cellular senescence pathway.

Nucleic acid, protein, and protein-bound lysine and arginine patterns in epidermal nuclei of the mature and senescing onion bulb.

Eight macromolecular indices were determined for nuclei of outer epidermal cells in the equatorial plane from young through old leaf bases (sequential leaf senescence) and for nuclei in basal, equatorial and three apical locations within one leaf base (apical cell senescence). The eight nuclear indices were total nucleic acid. DNA, RNA, histone, non-histone, total protein, and protein-bound lysine and arginine. Cell senescence and death were accompanied by startling decreases in these eight indices. In sequential leaf senescence, nuclei in the second innermost turgid leaf base contained about four to five times the amount of nucleic acids and nuclear proteins in the youngest leaf base. The nuclei in the outermost leaf base contained about 20% less of the nucleic acids and proteins measured than the second outermost leaf base. We infer that this represents a decline in nuclear constituents due to senescence, since less than 30% of the cells in the innermost paperlike leaf base contained nuclei and those present were almost devoid of the nuclear constituents measured. Cell senescence in the apical section of a turgid leaf base showed sequential declines in all nuclear indices studied from areas composed of living cells to areas at the apex composed of dead cells. Possible changes in histone content due to physiological ages were detected in the equatorial but not in the vertical study; the protein-bound lysine to arginine ratios increased from younger to older leaf bases.

Effect of ethylene on selective ribosomal cistron regulation in quiescent and senescent onion leaf base tissue

Excised pieces of tissue from four regions (base, equator, near-apex, and apex) of yellow, sweet Spanish onion bulbs were treated with ethylene in an attempt to activate minor nucleolar organizer regions (NORs). In the quiescent equatorial and near-apical regions, major nucleoli in control and ethylene-treated tissue increased in size and changed morphologies. The minor NORs were expressed only in the ethylene-treated tissue. These data confirm and extend our previous findings. In the quiescent basal region, major nucleoli increased in size and changed morphology from round to oval, elongated-oval and dumbbell shapes in both control and ethylene-treated tissue. However, the number of elongated-oval and dumbbell types were one-third that observed in equatorial and near-apical tissue. Minor NORs were not expressed. We infer that the responses of the major nucleoli and the inability to activate minor NORs were due to hormone interactions that affect the expression of rRNA cistrons (methylation-demethylation of CpG sites in the external spacers) in this basal (meristematic) region. In the senescing apical region, major nucleoli in control and ethylene-treated tissue did not change in size or morphology and ethylene had no effect on minor NORs. We infer that the cells in the apical region had passed the point of no return in the senescence pathway. We propose that the inability to activate major and minor NORs in senescing tissue is one of the earliest symptoms of cellular senescence and is associated with karyoskeleton degeneration.

Experimental Retracement of Terrestrial Origin of an Excitable Cell: Was it Predictable?

A largely historical treatment of how the evolutionary sequence to the first synthesis of life under terrestrial conditions, as retraced in the laboratory, is presented. The insights of such pioneers as Pasteur on self-organization of matter to “beings” are related. The recent acknowledgment of fruitless attempts by experts in DNA-first. RNA-first. and in irrelevance of the concept of random beginnings are cited. Updating of origins from amino acid-instructed genetically primary thermal protein is detailed. The essence of the finding of internal nonrandomness is emphasized, as well as the critical nature of studying the initial steps of molecular evolution in a synthetic direction. The total flowsheet for amino acid precursors ➔ protocell = protoneuron is updated. The results of experiments are tested against the characteristics of life as it is usually defined: metabolism, growth, reproduction, and response to stimuli such as light.

Ethylene is a selective ribosomal cistron regulator in Allium cepa epidermal cells

In Allium cepa L. (onion) the number, size, and morphology of visible nucleoli per nucleus varies during cell division, growth, differentiation, storage, activation of quiescent tissue, senescence, wounding, and disease (host-pathogen interactions). Since there are two types (differentiated visually by size) of ribosomal cistrons in onion (major and minor nucleoli differ in the external spacer nucleotide sequences), we inferred that they function separately. In controls, major nucleolar organizer regions (NORs) were visible in epidermal cell nuclei as nucleoli. After exposure of the tissue to ambient conditions, these nucleoli enlarged and changed morphology. Minor NORs (when in the genome) required more than 6 h to become developed as visible nucleoli. In the ethylene treatments, the major NORs (visible nucleoli in quiescent epidermal cells) increased in size a (activation of a greater number of tandem rRNA genes) as in the controls. The minor, quiescent NORs became active and visible (activation of quiescent, inactive NORs) within 3 h. Actinomycin D, and cycloheximide, with or without ethylene, inhibited the increase in size of the major NORs, and prevented activation of the minor NORs (i.e. they did not become visible). Silver nitrate and cobalt chloride had no effect on major NORs but inhibited the appearance of minor nucleoli when combined with ethylene treatments. We infer: ethylene acts to regulate the expression of the minor NORs (selective ribosomal cistron regulation); and, other hormone(s) are involved in the regulation of the major NORs. Yellow and white sweet Spanish onions we studied had two major NORs and two minor NORs. Red sweet Spanish onions we studied had only the two major NORs.

Effect on germination and post-germination development of Colletotrichum dematium var circinans due to light and dark incubation and coverslip placement

Light regime was not a major factor in germination of spores of Colletotrichum dematium var circinans. Number of germ-tubes and appressoria formed were affected by incubation regime and coverslip placement. Most germ-tubes and appressoria were produced on spores under shimmed coverslips. Light incubation favored germ-tube production and dark incubation favored appressoria production. Predicted time to 100% germination was affected by treatments. Shortest times were predicted for light incubated spores under shimmed coverslips. Longest times were predicted for non-coverslipped spores. A significant number of appressoria were produced sessile on spores. Most sessile appressoria were produced on dark incubated spores. Approximately 56 % of all spores germinated, and approximately 60% of all germ-tubes and appressoria were produced on these spores which were situated closest to coverslip or droplet edges. Placement of germ-tubes on spores was not affected by treatments. However, percentages of germ-tubes, initiated from various areas of spores, were significantly different. This variation appears to be due to factors internal to spores.

Changes in nuclear macromolecules and nucleolar volume associated with selective ribosomal cistron activation by ethylene

Pieces of leaf base tissue from the equatorial region of the third turgid onion (Allium cepa L.; yellow, sweet Spanish, quiescent tissue) leaf base with two visible major nucleoli (volume = 0.51 microns3) and two inactive minor nucleolar organizer regions (NORs) in the outer epidermal cells were placed in shallow water (time = T0 control). After 3 h (T3 control), the two visible nucleoli enlarged (178% of T0) and changed shape (from 88% round and 12% oval at T0 to 5% round, 68% oval, 20% elongated-oval, and 7% dumbbell). The minor NORs remained inactive. Nuclear RNA and non-histone protein (nHP) increased (RNA = 128% and nHP = 134%, compared with T0 data, set at 100%). The content of DNA and histone protein (H) in nuclei remained unchanged. Major nucleoli in pieces of onion placed in 10(-5) M ethylene enlarged and minor nucleoli became visible in 33% of the nuclei; i.e., ethylene acted as a selective ribosomal cistron regulator. The nucleolar volume (212% of T0) was statistically greater than those for T0 and T3. DNA and H remained unchanged (not statistically different) but RNA (159% of T0) and nHP (169% of T0) were greater (statistically significant) than those for T0 and T3 controls. Cobalt chloride (10(-5) M Co) alone or in 10(-5) M ethylene (Co + E) inhibited the enlargement of some of the major nucleoli and completely inhibited the appearance of minor nucleoli. The nucleolar volumes for the cobalt and for cobalt-ethylene treatments were significantly less than those for T3 controls and ethylene treatment at T3. DNA and histone did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in nucleic acid and protein content in nuclei of human cervical cells

Nuclei in five classes of cervical cells observed in Pap smears were studied using quantitative epifluorescence microscopy. The five classes of cells were: parabasal (Pb) cells; intermediate cells with round (I-R), oval (I-O), and rod-pyknotic (I-RP) nuclei; and, pyknotic (P) cells. Six nuclear traits were measured: total nucleic acid, DNA, RNA, total protein, histone, and non-histone protein. The six nuclear indices increased as Pb cells became I-R cells (cell enlargement and maturation), and then decreased as I-R cells degenerated through the following senescence sequence: I-O----I-RP----P. We infer that these changes continue and result in anucleate, superficial cells. Pb cells are probably in early stages of DNA synthesis (S-phase of the cell cycle) since the mean for DNA increased as they became I-R cells. The following types of cells comprised the Pap smears studied: Pb, 7%; I-R, 19%; I-O, 55%; I-RP, 8%; P, 9%; superficial cells with nuclei devoid of nucleic acids, 1%; and, anucleate cells, 1%. We conclude that cervical exfoliative cytology provides a model system for the study of human cell development, maturation, senescence, and death in addition to its use in detecting early through late stages of cervical cancer. The high correlation between the nuclear indices studied suggests that several quantitative nuclear parameters other than DNA may be useful for cancer detection.

Germination and post-germination development of Colletotrichum dematium f. circinans on Allium cepa

Spores of Colletotrichum dematium f. circinans were inoculated onto the abaxial epidermis of washed and unwashed pieces of leaf-base tissue taken from the first thru fourth layers of white, yellow and red onions. Washing tissue dramatically improved germination, less than 0.1% for unwashed versus 83% for washed. Germination capacity, and the number of germination types (16) found on washed tissue was not affected by onion bulb color or leaf-base layer inoculated. Spores with one sessile appressorium, type 001, or with one germ tube without an appressorium, type 100, or with one germ tube with an appressorium produced at the tip, type 110, made up approximately 93% of the types for those spores which germinated.