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A M Zimmerman

Publications and source records attributed to A M Zimmerman.

At least 37 records · Page 2Linked to original sources
PubMed1987

Cell cycle changes in water properties in sea urchin eggs.

This study concerned changes in the motional properties of cellular water during the first cell cycle of fertilized sea urchin eggs (Lytechinus variegatus). There was a significant decrease in proton NMR T1 relaxation time and in cytoplasmic ice crystal growth during mitosis and a significant increase in T1 time and cytoplasmic ice crystal size during cleavage. This was not caused by egg water content changes as reflected by egg volume measurements. Removal of both the fertilization membrane and the hyaline layer shortly after fertilization did not alter the pattern of T1 time changes at mitosis and cleavage as compared to whole eggs; thus, the pattern of T1 time changes was attributed to intracellular events. Treatment of fertilized eggs with cytochalasin B, an inhibitor of actin polymerization, did not block the fall in T1 time at mitosis, but did block cytokinesis and the increase in T1 time, which normally occurred at cleavage. A significant pattern of actin disassembly and reassembly at mitosis and cytokinesis was found by studies on the total amount of monomeric actin (G actin) using the DNase I assay. This led to the hypothesis that the observed changes in T1 time and ice crystal size during the first cell cycle were due to the depolymerization and polymerization of cytoplasmic actin. To test this, the effect of the in vitro polymerization of purified actin on the T1 time and on ice crystal growth was examined. It was concluded that changes in the T1 time and ice crystal growth upon polymerization of actin in vitro resembled the changes seen in vivo. These results suggest that changes in the motional properties of cytoplasmic water during the first cell cycle are due, at least in part, to the state of polymerization of cytoplasmic actin.

Actins↗
PubMed1987

Effects of cytoskeletal inhibitors on water proton relaxation time changes in unfertilized and fertilized sea urchin eggs.

Unfertilized and fertilized sea urchin eggs were used for pulsed proton NMR spin-lattice relaxation time (T1) measurements of cellular water. An 81% increase in T1 time at fertilization was largely explained by the accumulation of extracellular water in the perivitelline space. To assess the role of microtubule and actin filament assembly and disassembly, eggs were treated with drugs that are known to change these cytoskeletal elements (i.e., colchicine, taxol and cytochalasin B). Egg volume was also monitored in all studies to rule out the influence of water content changes on the observed T1 relaxation time changes. Neither assembly nor disassembly of microtubules changed the T1 relaxation time. The role of actin polymerization and depolymerization is discussed as a possible explanation for the observed cell cycle dependent water proton T1 relaxation time changes.

Actins↗
PubMed1987

Cannabinoids reduce fertility of sea urchin sperm.

Cannabinoids are potent pharmacological substances derived from marihuana. The effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) on fertilization in the sea urchin Strongylocentrotus purpuratus were investigated. Insemination of THC-treated eggs (5-400 microM) with excess sperm did not result in polyspermic fertilization. At minimal sperm densities, THC (0.1-10 microM) inhibited fertilization in a dose-dependent manner. Pretreatment of eggs with THC did not reduce their receptivity to sperm. Pretreatment of sperm with THC reduced their fertilizing capacity. The concentration of THC required to reduce sperm fertility by 50% was 1.1 +/- 1.1 microM. The fertilizing capacity of THC-treated sperm depended on concentration of sperm and duration of pretreatment. The fertility of sperm at minimal densities was reduced by 50% at 129.3 +/- 43 s treatment with 10 microM THC. The adverse effect of THC on sperm fertility was reversible. CBN and CBD at comparable concentrations (0.1-10 microM) inhibited fertilization in a manner similar to THC. First division was not delayed in zygotes that were fertilized with sperm pretreated with 10 microM THC. These studies show that cannabinoids directly affect the process of fertilization in sea urchins by reducing the fertilizing capacity of sperm.

Animals↗
PubMed1986

Effect of cannabinoids on spermatogenesis in vivo: a cytological study.

The cytogenetic effects of delta 9-tetrahydrocannabinol (THC) and cannabinol (CBN) (10 mg/kg) were investigated in hybrid mice of genotype (C57BL x C3H)F1. Mice were treated for 5 consecutive days with the specific cannabinoid; 16 days after the last treatment the meiotic cells were evaluated. Analysis of the spermatocyte bivalents at the first meiotic metaphase failed to reveal any numerical or structural abnormality. Contrary to previous reports we failed to find any major meiotic abnormalities associated with THC and CBN treatments. There was no evidence of ring or chain figures. The centromeric banding procedure (C banding) was used to support observations of Giemsa stained cells. It has previously been reported that cannabinoids suppress the incorporation of 3H-uridine into pachytene spermatocytes and round spermatids; the absence of chromosome aberrations in the present study suggests that an adverse effect appears to be present, but may be at a level other than cytogenetic.

Animals↗
PubMed1985

Actin and tubulin in Tetrahymena.

Tubulin and actin are cytoskeletal proteins known to play a major role in dividing cells. Tetrahymena pyriformis, a ciliated protozoan, was used as a model system for investigating tubulin synthesis during cilia regeneration and during the cell cycle. Until recently the identification of actin in Tetrahymena has been controversial. In this report evidence for the presence of actin in Tetrahymena is reviewed and control of actin gene expression during the cell cycle is discussed.

Actins↗
PubMed1985

Biochemical evidence for the presence of an actin protein in Tetrahymena pyriformis.

A protein from an ATP extract prepared from an acetone powder of Tetrahymena pyriformis GL was identified as actin. The protein migrated slightly behind muscle actin on sodium dodecyl sulphate (SDS)/10% polyacrylamide gels (SDS/PAGE) with an apparent molecular weight of 47 500 (47.5 X 10(3) Mr). Partial proteolysis of this band with Staphylococcus aureus V-8 protease followed by electrophoresis revealed a pattern of peptides in which at least four peptides were similar to those observed after digestion of rabbit skeletal muscle actin. The 47.5 X 10(3) Mr protein appeared particularly susceptible to endogenous proteolytic cleavage, which was inhibited by leupeptin. An ATP extract prepared with leupeptin was applied to a DNase I-affinity column and a distinct peak was eluted with 3 M-guanidine . HCl; the DNase I-binding protein appeared as a distinct band on SDS/PAGE with an apparent molecular weight of 47.5 X 10(3) Mr. In the absence of leupeptin, the DNase I-binding protein appeared as a broad 34 X 10(3) Mr band on gels. Both the ATP extract and the DNase I-binding protein showed reactivity with commercially available antiserum raised against native chicken skeletal muscle actin as determined by an enzyme-linked immunosorbance assay (ELISA). Immuno-blotting studies and affinity purification of this antiserum showed that the recognition was not specific to the 47.5 X 10(3) Mr protein. However, using affinity-purified anti-actin antibodies raised against denatured actin from chick smooth muscle, recognition of the 47.5 X 10(3) Mr protein and a 34 X 10(3) Mr protein was shown. In negatively stained preparations from an ATP extract after two cycles of polymerization and depolymerization there were filaments, 8-12 nm diameter, which did not decorate with subfragment S-1 of myosin, but which resembled intermediate filaments. Analysis of these filaments on SDS/PAGE indicated an intensely stained 54 X 10(3) Mr band. It is suggested that, in vitro, Tetrahymena intermediate filaments assemble under conditions expected to assemble actin filaments. Thus, in Tetrahymena there is a protein that resembles actin in its extractability, molecular weight, peptide pattern after partial proteolysis, DNase I-binding capacity and reactivity with anti-actin antibodies. However, this protein did not assemble into actin filaments in crude extracts.

Actins↗
PubMed1985

Water ordering during the cell cycle: nuclear magnetic resonance studies of the sea-urchin egg.

Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by nonmicrotubular factor(s) of the metaphase egg.

Animals↗
PubMed1984

Abundance of tubulin mRNA on polysomes following deciliation and during the synchronous cell cycle of Tetrahymena.

The protozoan, Tetrahymena pyriformis GL, was used as a model system for studying polysomal mRNA during the cell cycle and during cilia regeneration. Our previous work has shown a substantial induction of tubulin synthesis following deciliation and during G2 of the synchronous cell cycle. In the present study, the abundance of tubulin mRNA on polysomes was examined in order to determine whether more tubulin mRNA was being translated during the periods of peak tubulin synthesis. Polysomes isolated at sequential times following deciliation and during the synchronous cell cycle were translated in a cell-free translation system derived from wheat germ. The abundance of tubulin mRNA on polysomes was inferred from the amount of tubulin translated in vitro. Following deciliation and prior to the peak period of tubulin synthesis, the abundance of tubulin mRNA (at 140 min post-deciliation) increases to 25 times the initial value observed (at 20 min post-deciliation). Since the increase in tubulin mRNA abundance precedes the peak in tubulin synthesis, induction of tubulin synthesis appears to be mRNA-dependent. A similar analysis of tubulin mRNA abundance on polysomes during the synchronous cell cycle revealed a peak of tubulin mRNA prior to each peak of tubulin synthesis. These studies suggest that the periodic fluctuations in the synthesis of tubulin are dependent upon fluctuating levels of tubulin mRNA on polysomes.

Animals↗
PubMed1984

Effects of cannabinoids on macromolecular synthesis in isolated spermatogenic cells.

Effects of delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) (1, 3 or 5 microM for 2 h) on macromolecular synthesis were investigated in homogeneous populations of pachytene spermatocytes and round spermatids obtained from CD-1 mice. Incorporation of 3H-uridine into the acid-insoluble fraction was used as an index of RNA synthesis. Treatment of pachytene spermatocytes with 5 microM THC, CBN and CBD reduced radioactive incorporation by 81%, 62% and 67%, respectively, compared to vehicle (dimethylsulfoxide) controls. Treatment of round spermatids with 5 microM THC, CBN and CBD reduced radioactive incorporation by 54%, 61% and 63%, respectively, compared to controls. In addition to reduction of radioactive incorporation into the acid-insoluble fraction, there was also reduction in the acid-soluble fraction not comparable in magnitude to the reduction in the acid-insoluble fraction. Pachytene spermatocytes and round spermatids isolated from THC-treated mice (10 and 20 mg/kg) showed no reduction in macromolecular synthesis compared to cells isolated from vehicle-treated mice. Suppression of macromolecular synthesis induced by in vitro cannabinoid treatment is discussed in terms of membrane-mediated interference with cell metabolism.

Animals↗
PubMed1984

Action of delta 9-tetrahydrocannabinol on the pool of acid soluble nucleotides.

The effects of delta 9-tetrahydrocannabinol (THC) treatment on acid soluble pools of uridine nucleoside and nucleotides were investigated in Tetrahymena pyriformis and in isolated mouse lymphocytes and spermatogenic cells. In THC treated Tetrahymena and mouse lymphocytes the uptake of labelled precursor into acid soluble pools of uridine nucleoside and nucleotides fluctuated, whereas in pachytene spermatocytes and round spermatid cells the labelled pool was reduced. The reduction in the labelled pool measured in mouse spermatogenic cells was attributed primarily to a reduction in radioactively labelled uridine nucleoside. Treatment of Tetrahymena in high concentrations of THC (960 and 3,200 microM) resulted in an increase of labelled uridine nucleoside and a reduction in the amount of labelled uridine nucleotides. Expansion of the acid soluble pool with radioactive uridine resulted in small differences in labelled nucleoside and nucleotides in control and THC treated Tetrahymena and mouse lymphocytes. The results are discussed in terms of the effects of THC on macromolecular synthesis in various cellular systems.

Animals↗
PubMed1984

Composition of the isolated mitotic apparatus: analysis by polyacrylamide gel electrophoresis.

The mitotic apparatus (MA) from zygotes of the sea urchin Strongylocentrotus purpuratus were isolated and the components of the isolated MA were studied using one-dimensional polyacrylamide gel electrophoresis. MA were isolated using two different stabilization media, hexylene glycol and glycerol-dimethylsulphoxide ( MTM ), because previous studies have shown that MA isolated with these solutions have different properties. Both MA have at least 15-20 bands on gels. Whereas a few bands might be unique to one method of isolation, most bands are the same in hexylene glycol MA and MTM MA. Both kinds of MA differ from the general cytoplasm. Detailed descriptions of the appearances of the gels are given. The main difference between the two kinds of MA is in the relative intensities of the bands found in both MA. The physical and chemical properties of MA change during storage. After various durations of storage MA were extracted with 0.5 M KCl: more proteins were extracted from freshly isolated MA than from stored MA. There were changes in extractibility which occurred at the same time as the physiological changes, but the particular changes in hexylene glycol MA were not the same as those in MTM MA. Thus, there is no common denominator that would allow us to say that loss of extractibility is associated with changes in a certain protein of the isolated MA.

Animals↗
PubMed1983

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle.

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.

Actins↗
PubMed1983

Effects of delta 9-tetrahydrocannabinol, cannabinol and cannabidiol on the immune system in mice. I. In vivo investigation of the primary and secondary immune response.

The effects of the cannabinoids delta 9-tetrahydrocannabinol (THC), cannabinol and cannabidiol on the primary humoral immune response, the secondary humoral immune response and the memory aspect of humoral immunity in response to sheep red blood cell (SRBC) immunization was investigated. Mice treated with THC (10 and 15 mg/kg) during the primary immunization period exhibited a suppression of the primary humoral immune response. Mice treated with THC during the secondary immunization period showed no measurable suppression of the secondary humoral immune response to the immunizing antigen. The memory aspect of humoral immunity was assessed when treatment with cannabinoids was carried out during the primary immunization period and the ability of mice to undergo a secondary immune response was evaluated; suppression of the secondary humoral immune response was evident with THC treatment (10 and 15 mg/kg). Cannabinol and cannabidiol (10 and 25 mg/kg) treated mice showed no impairment in the ability to undergo primary or secondary immune responses with any treatment protocol. In vivo investigations of the effects of cannabinoids on the thymus were also carried out. Thymus weight and thymus cell number were depressed in mice undergoing a primary humoral immune response when treated with THC (10 and 15 mg/kg) during this period. THC treatment, however, did not alter these parameters in mice not challenged with antigen. In both challenged and unchallenged animals, cannabinol and cannabidiol did not measurably alter the thymus.

Animals↗
PubMed1983

Effects of delta 9-tetrahydrocannabinol, cannabinol and cannabidiol on the immune system in mice. II. In vitro investigation using cultured mouse splenocytes.

The effects of the cannabinoids, delta 9-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on the primary-like immune response were investigated in primary cultures of mouse splenocytes. Splenocyte cultures were stimulated with sheep erythrocytes in vitro and incubated with cannabinoids for the first 24 h after antigenic stimulation (prior to initiation of DNA synthesis), from 24 h to 6 days after antigenic stimulation, and for the entire 5-day period. THC (1 and 5 microM) and CBD (5 microM) depressed the primary-like immune response of stimulated mouse splenocytes when incubated for the first 24 h after antigenic stimulation and the entire 6-day culture period. CBN did not show any measurable suppression of the primary-like immune response. Treatment of splenocyte cultures with cannabinoids after the first 24 h after antigenic stimulation showed no impairment of the in vitro primary-like immune response.

Animals↗
PubMed1983

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena.

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.

Animals↗
PubMed1981

Effect of delta 9-tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena.

Cyclic nucleotide levels were determined in division-synchronized Tetrahymena and the effect of delta 9-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non-drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of cAMP was statistically significant; however the variation in the level of cGMP was not statistically significant. THC caused a lowering of cAMP and cGMP levels throughout the 4-experimental treatment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8-15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (110-120 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is discussed in relation to delays in the division schedule.

Animals↗
PubMed1980

Experimental determinations of tubulin in the in vivo mitotic apparatus of sea urchin zygotes.

Mitotic apparatus (MA) were isolated from zygotes of a sea urchin (Strongylocentrotus purpuratus), using hexylene glycol (pH 6.4) as lysing-stabilizing agent. Protein was measured in the MA pellet and in the remainder of the cell lysate (using the Lowry procedure). Tubulin was measured in the MA pellet and in the remainder of the cell lysate (using microdensitometry of stained gels after sodium dodecyl sulphate - polyacrylamide gel electrophoresis). From these data we calculated the maximum possible amounts of tubulin in the isolated MA and in the MA in vivo; in these calculations we assumed that all the tubulin in the cell is associated with the MA, and we assumed that, as reported in the literature, the MA lose 90% of their dry matter during the isolation. We conclude that tubulin probably comprises less than 7% of the protein in the in vivo MA, and, even if there are very large errors, tubulin is considerably less than half the protein in the MA.

Animals↗