Diploid gynogenesis induced by hydrostatic pressure in rainbow trout, Salmo gairdneri Richardson

Diploid gynogenetic fry of rainbow trout, Salmo gairdneri , were produced by treating eggs activated by UV-irradiated sperm with hydrostatic pressure. Treatments for l0 min at 40 min after activation with 55 000 kPa (8000 psi) pressure resulted in high hatching rates and yields of heterozygous diploid gynogenomes of up to 81%. Attempts to produce homo-zygous diploid gynogenomes by the suppression of the first mitotic division ofthe eggs failed. Ether treatment alone did not induce diploid gynogenesis.     

Salinity and oxygen tolerances of eggs and larvae of Hawaiian striped mullet,Mugil cephalus L.*

Salinity and oxygen tolerances of eggs and larvae of Hawaiian striped mullet (Mugil cephalus L.) were studied. Optimal salinities for eggs incubated at 19.5-20.5°C were 30–32‰. Maximum larval survival occurred within the salinity range of 26–28‰. Significant decreases in egg survival occurred with eggs incubated in mean oxygen concentrations below 5.0 p.p.m. and for larvae held in mean oxygen concentrations below 5.4 p.p.m. The results are discussed in terms of spawning sites and the effects of oxygen saturation values on fish eggs and larvae.     

EFFECTS OF MICROTUBULE INHIBITORS ON PRONUCLEAR MIGRATION AND EMBRYOGENESIS IN FUCUS DISTICHUS(PHAEOPHYTA)1

Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed.     

Changes in mitotic indices in roots of Vicia faba

Roots of Vicia faba were treated with solutions of colchicine or IAA or both. Mitotic indices and the frequencies of the different stages of mitosis were determined immediately after a three hour treatment or following a 24 hour period of recovery. Roots scored after treatment with colchicine for three hours showed several effects, none of which were reversed by simultaneous treatment with IAA. Treatment with IAA for three hours had little detectable effect on mitotic index (MI) on the frequencies of the various stages of mitosis. After a recovery period, following a three hour treatment, of 24 hours, colchicine treated roots showed a significant increase in their MI; this was due largely to an increase in the number of metaphases but it was also due in part to the presence of tetraploid cells in division. IAA treated roots revealed an inhibition of mitotic activity, which was most marked at 3.13–6.26×10^–4 M IAA. The results from roots treated with mixtures of colchicine and IAA for three hours and fixed 24 hours later showed: 1) the increase in MI induced by colchicine is reversed by IAA, the intensity of the reversal increasing with increasing concentrations of IAA; 2) reductions in the total numbers of cells in prophase or in metaphase occur after treatment with different concentrations of IAA; 3) IAA leads to a reduction in the number of tetraploid cells seen in division.It appears that colchicine induces a change in the pattern of mitotic activity 24 hours after the end of treatment and its effects are reversed by IAA. At 4.2×10^–4 M IAA a balance occurs between the opposing effects of colchicine and IAA and the MI is not significantly different from that of the controls. It is suggested that one result of a treatment with colchicine is a change in the level of growth factors in root meristems. This change, which appears to result in a temporary increase in MI is reversed by the addition of IAA. Thus one of the growth factors, the level of which has been affected, is replaceable by exogenous IAA.     

Comparison of the Disruption of Mitosis and Cell Plate Formation in Oat Roots by DCPA, Colchicine and Propham

Holmsen, J. D. and Hess, F. D. 1985. Comparison of the disruptionof mitosis and cell plate formation in oat roots by DCPA, colchicineand propham.—J. exp. Bot. 36: 1504–1513. Concentrationsof DCPA, propham and colchicine were selected to cause from0% to greater than 60% inhibition of oat (Avena sativa L. ‘Victory’)root growth after 24 h exposure. Root growth progressively declinedas concentrations were raised from 1·0 to 5·6mmol m^–3 DCPA, 1·0–5·0 mmol m^–3propham, and 50–500 mmol m^–3 colchicine. In additionto inhibiting root growth each mitotic disrupter caused theroot tips to swell to an extent dependent upon concentration.All three compounds effectively disrupted mitosis at concentrationsthat caused less than maximal root growth inhibition. Mitoticdisruption was manifest as a reduction in the number of normalmitotic figures and an increase in the number of condensed prophase,multipolar and anaphase bridge division figures. The frequencyof each type of division figure was different for each of thethree compounds. DCPA disrupted mitosis more effectively whencompared with propham and colchicine at concentrations whichcaused the same amount of root growth inhibition. Each mitoticdisrupter also induced the formation of aberrant cell walls.DCPA was the most effective at disrupting cell plate formation,whereas colchicine was least effective. These data suggest thatthe mechanism of action of DCPA is distinct from the mechanismof colchicine or propham Key words: Avena sativa L., mitotic disruption, DCPA, colchicine, propham     

CELL DIVISION STUDIES OF THE SHOOT APEX OF DATURA STRAMONIUM DURING TRANSITION TO FLOWERING

Seedlings of Datura stramonium L., although not photoperiodically sensitive, are useful for floral transition studies when raised in a growth chamber at a constant temperature of 25 C with a photoperiod of 8 hr of light (1,600-2,000 ft-c) and 16 hr of darkness. A terminal flower is formed after the seventh or eighth leaf primordium is produced. A constant rate of leaf initiation up to the time of flowering enables specific apical stages to be obtained and studied. Changes in the mitotic index, substantiated with calculated rates of cell division (measured by the accumulation of metaphases following treatment with colchicine) were studied in shoot apical zones during transition to flowering. Fluctuations in the mitotic index of each zone in the vegetative and transition apex with respect to apical stage as well as time of day were not statistically significant. The mitotic index of the summit zone of the vegetative apex was significantly lower than in the other zones whose mitotic indices were not significantly different from one another. During floral transition the mitotic index of the summit zone as well as the central zone (just below the summit zone) significantly increased while no significant changes were detected in the flank zones. It was shown that the mitotic index could be considered representative of the rates of cell division in Datura.     

Cucurbitacine als stathmokinetische Wirkstoffe

Summary A synergism of colchicine and different cucurbitacins has been shown, to be a cause in the formation of ball-metaphases in Bryonia species. The bitter principles cucurbitacin L and I, dihydrocucurbitacin B, and tetrahydrocucurbitacin I, which were examined more closely with respect to their karyological effects, were shown to give rise to C-metaphases in Hordeum and Vicia at concentrations of about 0.016 per cent. On the one hand the number of mitotic stages is reduced, and on the other hand there is an increase of metaphase stages. Higher concentrations lead to pycnotic degenerations of nuclei and are toxic. When colchicine is applied together with cucurbitacins, ball-metaphases result. This could be demonstrated in plants lacking colchicine and cucurbitacins (Hordeum) when both substances were applied at the same time. Ball-metaphases also appear in plants containing cucurbitacins (Bryonia alba, B. dioica, Citrullus colocythis, Iberis amara) when they are treated with colchicine.     

Okadaic acid inhibits sister chromatid separation in mammalian cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid inhibiting phosphatase 2A activity alone or in addition to phosphatase 1 activity. Phosphatase 2A inhibition alone had no visible effect on mitosis, but inhibition of both phosphatase 1 and 2A produced mitotic abnormalities, including inhibition of anaphase mimicking the effect of colchicine. Recovery experiments in okadaic acid-free medium showed formation of diplochromosomes, indicating a failure of sister chromatid separation in the treated mitotic cells. The universality of the phosphatase 1 requirement in sister chromatid separation is discussed.     

Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

Potential for high hydrostatic pressure processing to control quarantine insects in fruit

Tests were conducted to determine the potential for high hydrostatic pressure (HPP) to control codling moth, Cydia pomonella (L.), and western cherry fruit fly, Rhagoletis indifferens Curran. Apples (Malus spp.) with codling moth larvae or eggs were treated at 24 and 72 h, respectively, after infestation at a series of pressures between 14,000 and 26,000 pounds per inch2 (psi). Survivorship was determined the next day for larvae and after 10 d for eggs. Codling moth eggs were more tolerant of HPP treatment than larvae. Mortality of larvae was 97% at 22,000 psi, whereas mortality of eggs at this dose was 29% and not significantly different from the untreated controls. In a second study, no codling moth eggs hatched at any high pressure treatment between 30,000 and 80,000 psi, indicating these pressures were lethal. Various stages of western cherry fruit fly were treated at pressures from 10,000 to 45,000 psi, and survivorship was determined after 24 h. Eggs and third instars were more tolerant of HPP than the first and second instars. Mortality was 100% in western cherry fruit fly eggs and larvae at pressures > or =25,000 psi. Apple and sweet cherry quality after high pressure treatment was poor, but high pressure may have applications to control quarantine pests in other fruits.     

Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.     

Induction of Tetraploid Gynogenesis in the European Sea Bass (Dicentrarchus labrax L.)

A preliminary study on tetraploid gynogenetic induction in the European sea bass was performed by pressure-blocking the second polar body release and the first cleavage in eggs fertilized with ultraviolet-irradiated sperm. Fertilization of eggs with genetically inactivated sperm produced only haploid development that terminated around hatching. Pressure treatments (8.500 psi for 2 min) applied at 6 and 65 min after fertilization (a.f.) produced variable levels (7–95%) of tetraploid larvae at hatching. A small proportion of mosaics (3.8n/4.2n) was also recorded.     

Evidence for selective effects of vanadium on adipose cell metabolism involving actions on cAMP-dependent protein kinase

The insulin-like effects of vanadiumin vivo are likely to be achieved at micromolar concentrations. Demonstrated effects of vanadium on adipose tissue of streptozotocin-diabetic rats include inhibition of basal and stimulated rates of lipolysis and effects on fat cell protein phosphorylation. The studies described below examined the effects of vanadium (to a maximum concentration of 0.5 mM) on adipose cells or tissuein vitro. Vanadium, added as a vanadyl-albumin complex or as sodium orthovanadate, produced a marked (greater than 50%) inhibition of isoproterenol-stimulated lipolysis. Inhibition of lipolysis equivalent to that seen with insulin, was achieved with 100 M vanadium. In contrast, no insulin-like stimulation ofde novo fatty acid biosynthesis was observed with vanadium below 0.5 mM. Surprisingly, the antilipolytic effects of vanadium persisted in the presence of cilostamide, an inhibitor of the insulin-sensitive isoform of cyclic nucleotide phosphodiesterase. Studies with purified preparations of the catalytic subunit of cyclic AMP-dependent protein kinase revealed dose-dependent inhibition with vanadyl-glutathione (to a maximum of 40% inhibition). Equivalent inhibition of cyclic AMP-dependent phosphorylation of Kemptide (50%) was observed upon incubation of freshly-prepared fat-pad supernatant fractions with vanadyl-glutathione. These results suggest that effects of low concentrations of vanadium may be mediated, at least in part, by actions on the catalytic subunit of cyclic AMP-dependent protein kinase.     

Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions

Fertilized sea urchin eggs undergo a series of rapid and synchronized mitotic divisions. Extracts were made at various times throughout the first three mitotic divisions and assayed for phosphorylating activity toward histone H1. Histone H1 kinase (HH1K) undergoes a transient activation (8- to 10-fold increase) 20 min before each cleavage. The amplitude of the HH1K peak strongly depends on the synchrony of the egg population. Concomitant cytological observations show that the time-course of HH1K correlates with the time-course of nuclear envelope breakdown and of metaphase. This correlation is observed at each cell division cycle. HH1K from each of the three first mitoses show identical time- and concentration-dependence curves as well as identical dose-inhibition curves with 6-dimethylaminopurine and quercetin, suggesting that the same (group of) kinase(s) is (are) activated before each cleavage. Ionophore A23187 does not trigger, but inhibits, HH1K activation; however, partial activation of the eggs with ammonia at pH 9.0 (but not at pH 8.0) triggers the transient HH1K activation. Appearance of the HH1K cycle requires protein synthesis since it is completely abolished in emetine-treated eggs. Although cytochalasin B blocks egg cleavage, it does not inhibit HH1K activation nor nuclear divisions. A prolonged HH1K activation cycle is observed in eggs arrested in metaphase with colchicine or nocodazole. Despite the existence of a cycle in cAMP concentration during mitosis, forskolin, an activator of adenylate cyclase, does not modify the time-course of HH1K activation and of cell division. The cycling HH1K is independent of calcium-calmodulin, calcium-phospholipids, or cyclic AMP. It clearly resembles the mammalian "growth-associated histone kinase." The relationship between the transient activation of HH1K and the intracellular mitotic factors driving the cell cycle is discussed.     

Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus,Gastropoda)

Summary The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3–1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5–50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100–200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs — as in untreated eggs — microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.     

The effect of adenosine-3':5'-cyclic monophosphate on the mitotic rate in regenerating Dugesia dorotocephala.

Regenerating posterior sections of the flatworm, Dugesia dorotocephala, were treated with varying concentrations of 5'-adenosine monophosphate (AMP), cyclic AMP (cAMP) and dibutyryl cyclic AMP (Bt2cAMP) for 24 h. M/2000 colchicine was added to the medium during the final 4 h of treatment to collect mitotic figures. The mitotic rate was significantly increased at 0.5, 0.1 and 0.01 mM concentrations of Bt2-cAMP. While Bt2cAMP and cAMP produced comparable results at 0.01 mM, only the Bt2-cAMP-treated organisms exhibited a significantly higher mitotic rate at the 0.1 mM concentration. Theophylline and sodium butyrate did not evoke any stimulatory effect on mitotic rate.     

Cell Division: Pressure-induced reversal of the antimeiotic effects of heavy water in the oocytes of the starfish,Asterias forbesi

In dermal melanocytes of Rana pipiens, colchicine is known to produce a gradual, dosage-dependent dispersion of melanin granules, irreversible over several hours. This effect is potentiated by a number of chemical agents that normally produce a reversible dispersion of granules. In the present study we examined the effect of high hydrostatic pressure on changes induced in melanocytes by colchicine. In Ringer's solution, samples of skin from a single frog were incubated for 30 minutes at room temperature with or without colchicine, 9 × 10^?5 M. Then two samples, one of which had been pretreated with colchicine, were successively subjected to 12,000 psi for one hour at 25 to 26°C. The degree of dispersion of melanin granules in melanocytes was observed before, during and after the period of pressure. In frog skin pretreated with colchicine, the usually gradual, irreversible dispersion of melanin granules in melanocytes was potentiated. Since high pressure is known to produce solational changes in protoplasm, such changes may accompany dispersion of melanin granules in melanocytes. If this be so, then sol-gel equilibria may be important in the action of dispersing and aggregating agents, many of which are hormones and other physiologically active agents. Finally, the present study supports the hypothesis that colchicine shifts protoplasmic sol-gel equilibria toward a less gelated condition.     

The effect of high hydrostatic pressure and low temperature on lactic dehydrogenase and glutamic oxalacetic transaminase

LDH and GOT can be used with assurance as indicators of pressure-temperature effects in most regions of interest, specifically below 20,000 psi. LDH was susceptible to pressure deactivation at pressure levels below those tolerated by chymotrypsin, trypsin and alpha-amylase of Bacillus subtilis (17, 18, 20). Samples of LDH and GOT cooled to −20 °C were deactivated to the greatest extent by the application of pressure. The presence of glycerine and DMSO appeared to increase the sensitivity of GOT and LDH to pressure deactivation. When pressure was applied before cooling all pressures above 15,000 psi resulted in some deactivation of LDH and all pressures above 20,000 psi resulted in some deactivation of GOT.     

Oxadiazole derivatives as a novel class of antimitotic agents: Synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines

Oxadiazole derivatives were synthesized and evaluated for their ability to inhibit tubulin polymerization and to cause mitotic arrest in tumor cells. The most potent compounds inhibited tubulin polymerization at concentrations below 1 microM. Lead analogs caused mitotic arrest of A431 human epidermoid cells and cells derived from multi-drug resistant tumors (10, EC(50)=7.8 nM). Competition for the colchicine binding site and pharmacokinetic properties of selected potent compounds were also investigated and are reported herein, along with structure-activity relationships for this novel series of antimitotic agents.     

THE MECHANISM OF COLCHICINE INHIBITION OF MITOSIS : I. Kinetics of Inhibition and the Binding of H3-Colchicine

H³-colchicine of high specific activity (2.5 curies per mM) was prepared in order to study the mechanism of colchicine inhibition of mitosis in cultures of human cells, strain K.B. No direct effects on the duration of the cell cycle or macromolecular synthesis were demonstrable at a concentration of colchicine which completely inhibited mitosis. The radioactive compound was bound to the cells at a rate proportional to colchicine concentration. The binding appeared to be reversible since the radioactivity of the cells reached a maximum value for a given concentration and was slowly lost after resuspension of the cells in fresh medium. A suitable exposure to colchicine produced accumulation of metaphase-blocked mitoses after the colchicine was removed from the medium. An exposure of 6 to 8 hours at 10^-7 M was sufficient to block essentially all the cells in metaphase, thus indicating that colchicine is bound to the majority of interphase cells. The data are in quantitative agreement with a mechanism involving reversible binding of colchicine to a set of cellular sites. Based on the correlation between the time of first appearance of blocked mitoses and the radioactivity per cell, it is suggested that if a critical fraction (3 to 5 per cent) of the sites are complexed, the cell is unable to form a functional mitotic spindle.