Pattern of DNA synthesis in nuclei of feeding and starving Amoeba proteus : A cytofluorometric study

The DNA content in isolated nuclei of Amoeba proteus was determined for each of the three groups of synchronized amoebae over different intervals after division. Several nuclei of each amoeba group were fixed 1 h after division, before the amoebae were fed. About h after division, some amoebae in each group were given food (Tetrahymena pyriformis), while the rest were left starving. Samples of the nuclei of fed and starved amoebae were fixed 24 h and (in different groups) 42–55 h after division. In each group from 22 to 48% of the fed amoebae had divided prior to the last nuclei fixation. Starved amoebae did not undergo division. In all three amoeba groups the nuclear DNA content of fed cells by the end of interphase had increased to 280–300% the value for 1 h amoebae. The nuclear DNA content of starved amoebae of all three groups was also increased, and in two groups it exceeded the initial level more than two-fold. However, in all three groups, it was lower than that of fed amoebae. In all the groups the nuclear DNA content in fed amoebae grew after 24 h, i.e. during the second half of interphase, the increase accounting for from 11 to 48% of the total increase. The hypothesis is put forward that the increase in the nuclear DNA content during the cell cycle of Amoeba proteus is the result of two processes: (1) one-time replication of the DNA of the whole genome; and (2) repeated replication of some part of the DNA. In amoebae the relation of the pattern of nuclear DNA synthesis to the diet is considered.     

Fasting does not abolish the diurnal oscillation of ornithine decarboxylase activity in Morris hepatoma 5123-C.

The activities of ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) were determined under conditions of feeding or fasting in the hepatomas and livers of rats bearing Morris hepatoma 5123-C. Prior to killing, the animals were entrained to a schedule of 12 hours of light followed by 12 hours of darkness with food (60% protein) available only during the first two hours of the dark period. With food available, ODC and TAT activities displayed diurnal oscillations in hepatomas and host livers, and in the livers of control (non-tumor bearing) animals, characterized by rapid increases in enzyme activity coincident with the onset of feeding followed by a decline to pre-feeding levels. When food was withheld the increase in ODC activity in host and control livers, and TAT activity in hepatoma, host and control livers was not evident. However, withholding food did not abolish the diurnal oscillation of ODC activity in hepatoma 5123-C.     

Microanalytical and preparative peptide separations with a modified technicon autoanalyzer

The techniques described in this paper have been used to obtain highly reproducible analytical and preparative peptide separations using small amounts of material (10,11).     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

A smaller molecular weight retinol binding protein in rat testis seminiferous tubules

In the cytosol fraction in rat testis seminiferous tubules a lower molecular weight protein of ～4,800 daltons that binds retinol with high specificity has been isolated and purified by ammonium sulfate precipitation and on Sephadex column chromatography. The hexane extract of the component gave a characteristic retinol fluorescence spectrum. The amino acid composition was qualitatively similar to the retinol binding protein in the blood with the exception that cystine and cysteine were absent.     

Hydration of peptides. I. Calculation of accessible surface areas for several conformations of a cyclic dipeptide

The concept of “static accessibility” to water has been used to determine the accessible surface area of a cyclic dipeptide: c(l-Thr-l-His). Different calculated and experimental conformations of this model molecule have been examined, which allows us to analyse the variations of accessibility of the hydration sites localized on the peptide backbone and on the polar side chains. The maximum solvation criterion involves a large destabilization of conformations governed by intramolecular interactions. The variations of the amphiphilic character with the conformations are relatively small. Nevertheless, the experimental conformation seems to reflect such a behaviour, especially in the crystal, in which the amphiphilic character is compatible with intermolecular interactions. The accessibility studies must be regarded only as a preliminary step to a more quantitative analysis of peptide hydration.     

Hydration of peptides. II. Determination of the preferred sites of interactions of a cyclic dipeptide with water

As a first step in the determination of the hydration scheme of small peptides, the hydration sites of the cyclic dipeptide c(l-Thr-l-His) have been determined by two empirical potential treatments. In the first approach the energy is calculated by using the “Caillet-Claverie's” potentials, including electrostatic, dispersion-repulsion and polarization contributions. In the second approach (EMPWI method), the energy is calculated by simplified treatment, taking into account the electrostatic interactions of a suitable charge distribution and the dispersion-repulsion contributions. In this study, only the crystalline conformation of the cyclic dipeptide is considered. The hydration sites determined can be classified in three groups: (a) bridging sites, in which water interacts with both side chains, (b) bridging sites in which water interacts with one side chain and the DKP ring, (c) individual sites in which water interacts only with one polar group. The agreement between the results obtained by the two calculations is sufficiently satisfactory. This allows us to use EMPWI potential for calculations of more complex systems.     

Depth-dependent reproductive output of the green sea urchin, Strongylocentrotus droebachiensis (O.F. Müller), in relation to the nature and availability of food

Reproductive output of green sea urchins (Strongylocentrotus droebachiensis O.F. Müller) in the field was highest at depths where preferred macro-algae were abundant, and lowest at depths where preferred macro-algae were overgrazed or replaced by non-preferred species (Agarum and Ptilota). Feeding rate and gonad indices of sea urchins in the laboratory were highest on a diet of preferred algae (Fucusdistichus L. subsp. edentatus (Pyl.) Powell, Laminaria longicruris Pyl., Desmarestia spp. and Saccorhizadermatodea (Pyl.) J. Ag.), and lowest on the less preferred Agarum cribrosum (Mert.) Bory, Ptilota serrata Kütz., and crustose corallines. Gamete production/unit area in overgrazed habitats was as great or greater than in kelp beds because of the higher biomass of urchins in overgrazed areas. Gonad weight and reproductive output of urchins from habitats poor in food can be increased by providing preferred foods.     

The effects of short- and long-term exposure of chick embryos to neutral red on the frequency of sister-chromatid exchange

The chick embryo was used to study the effects of neutral red (NR) on the frequency of sister-chromatid exchange (SCE) in specific tissues exposed to this mutagen for short and long periods as development proceeded. In short-term trials, aqueous NR at doses of 10, 25 and 100 μg was injected in 3-day and 6-day embryos. In each case, embryos were also treated with 5-bromodeoxyuridine (BrdU) for a 24-h period (two cell cycles) and harvested at 4 days and 7 days, resp. A long-term exposure (about 8 cell cycles) was achieved by exposing embryos to NR from day 3 to day 7 of incubation. At a NR dose of 25 μg, the chronic exposure resulted in a doubling of the rate of SCE (11.4/cell) over that observed in embryos exposed for only 24 h at either days 3–4 (6.0/cell) or days 6–7 (6.0/cell). At 100 μg of NR, the same relationship held with SCE rates of 14.2/cell for the chronic exposure versus rates of 8.0/cell (3–4 days) and 6.9/cell (6–7 days). At 10 μg of NR, no such accumulation of SCE occurred upon long-term treatment.These results show an enhanced SCE response upon growth of embryonic cells in the presence of NR for several days. This may be the result of the persistence of past lesions with the addition of more lesions upon continued exposure to NR.     

Hemoglobin synthesis in isolated erythroid colonies from the chick embryo.

Primary cultures derived from mechanically dissociated definitive streak chick blastoderms were grown in a warm air stream on the stage of inverted phase microscope, through which in vitro erythroid development could be observed. Proerythroid cells divide three or four times in 48 hr to give rise to erythroid colonies ranging from 10 to 1000 cells, depending on the size of the blastoderm fragments from which they were derived.Erythroid cell development follows a similar course in cultures grown in a carbon dioxide incubator. Colonies consisting of about 50 cells, derived from blastoderm fragments containing 5 to 10 cells, were isolated and labeled with [³H]leucine, and their labeled hemoglobins were analyzed by isoelectric focusing. Both early hemoglobins (E,M,P,P′, and P″) and late hemoglobins (A and D) are made in colonies derived from single blastoderm fragments. The ratio of late to early hemoglobins is about 1.7 in all colonies analyzed. The implications of this finding for the clonal model of erythroid development are discussed.     

High increment of triglycerols with ether linkages in the retina during anoxia

In the bovine retina incubated during 90 min in a medium lacking glucose without preoxygenation and gassed with O₂-free N₂ a striking rise in triglycerides with ether bonds was found. NaF and 2,4-DNP decrease the production of these molecules while cycloheximide (1.78mM) completely abolishes the phenomenon. The free fatty acid pool increases about six-fold and this rise is not affected by the inhibitors. Bovine serum albumin stimulates the free fatty acid and triglycerols production. The amount of these lipids decrease by further incubation under normoxia in the presence of glucose.     

Quantification of acetylcholinesterase,acid and alkaline phosphatases in the central nervous system of Schizodactylus monstrosus, D. (schizodactylidae:orthoptera). Effect of topical application of the pyrethrum

The newly emerged adult male and female Schizodactylus monstrosus D. were treated with an insecticide, pyrethrum or with petroleum ether in the case of controls. At selected intervals of 6 h, 12 h and 18 h after treatment, the brain and ventral nerve cord and ganglia were homogenized to estimate the activity of acetyl-cholinesterase, and acid and alkaline phosphatase. The resultant activities of these three enzymes showed that: acetylcholinesterase decreased rapidly, and acid and alkaline phosphatase increased significantly after pyrethrum treatment in both brain and ventral nerve cord with ganglia compared with the controls. The activities of alkaline phosphatase showed a marked fluctuation at different post-treatment periods in both the tissues. The results have been discussed in relation to the impact caused by the treated insecticide and its metabolism.     

A functional analysis on isolated rat islets of Langerhans: effects of dimethylsulfoxide and low-temperature preservation

Cryopreservation of pancreatic islets of Langerhans offers the possibility of storage of sufficient quantities of this tissue for transplantation in the treatment of certain forms of diabetes, as well as providing a means of precise histocompatibility matching. In these studies, the effects of dimethylsulfoxide and various cooling rates on islet function are examined. These studies demonstrate that islets treated with 1.4 M dimethylsulfoxide and slowly cooled at a rate of 0.3 degrees C/min release insulin biphasically upon glucose challenge. In addition, this stimulated release is significantly improved (P less than 0.05) by increasing the duration of post-thaw culture. After thawing, these cryopreserved islets also retain the capacity to synthesize insulin. Islets frozen at faster cooling rates (3, 14, and 48 degrees C/min) exhibit varying degrees of glucose-induced insulin release, indicative of freeze-induced damage. These manifestations of freeze-induced damage include high basal (nonstimulatory) insulin release rates, little or no increase in the stimulated rate versus the nonstimulated rate, and failure of the stimulated release to return to basal levels when the glucose concentration is reduced.     

The cryopreservation of Chlamydomonas.

A cryophilic strain of the unicellular green alga Chlamydomonas, C. nivalis was found to be more resistant to the stresses both of freezing and thawing and of shrinkage and rehydration than was a mesophilic strain C. reinhardii. C. nivalis was found to have a higher degree of unsaturation of phospholipid fatty acids. Following freezing and thawing of C. reinhardii there was a direct correlation between reduction in cell viability and loss of membrane selective permeability. Activation of intracellular phospholipases occurred at an early stage of freezing injury. Attempts to cold harden C. reinhardii were unsuccessful. For C. reinhardii methanol was the only effective cryoprotectant for freezing to and thawing from ?196 °C and the effects of cooling rate upon cellular survival are presented.     

Plasma androstenedione after injections of dexamethasone and luteinizing hormone releasing hormone in young post-pubertal bulls

This study was performed on six French Friesian bulls (aged 12 months at the onset of the experiment) on two separate occasions, 3 months apart and lasting 2 days each time. On each of both occasions, three bulls were submitted on the first day to an i.m. injection of dexamethasone (DXM, 20 mg) 6 h priot to a Luteinizing Hormone Releasing Hormone (LH-RH) challenge (0.250 mg i.m.) and the three others (control animals) to LH-RH only. On the second day, they all received a single LH-RH injection. The treated animals then served as controls on the second occasion and the controls as DXM—LH-RH treated individuals. Blood samples were taken at hourly intervals before LH-RH treatment (for 5 h), then every 15 min (for 2.5 h), and afterwards, every hour for 3 h.Androstenedione concentrations were significantly enhanced after LH-RH challenge in a similar manner on days 1 and 2 within groups, but the relative magnitude of the response in terms of area under the curve (ng/ml of plasma × 150 min) was lower in the DXM—LH-RH treated group than in the controls. In addition, the individual correlation between the testosterone and androstenedione responses was significant (P <0.05) in those animals treated but not in the controls. This study therefore suggests evidence of a double origin for androstenedione secretion, from the testes and probably from the adrenals.     

Alterations in the levels of deoxyuridine triphosphatase, uracil-DNA glycosylase and AP endonuclease during the cell cycle

Deoxyuridine triphosphatase and uracil-DNA glycosylase were assayed in K12 cells, a Chinese hamster temperature-sensitive mutant known to arrest in G1 when shifted to the non-permissive temperature. Stimulation of cell division resulted in an increase of both enzyme activities. Levels of both enzymes were minimal during G0 and maximal in S phase. By contrast, apurinic/apyrimidinic site endonuclease activity, which incises the DNA at the apyrimidinic site generated by the glycosylase, was constant throughout the cell cycle.     

Binding of monoclonal antibodies to the nuclear estrogen receptor in intact nuclei

A monoclonal antibody to calf uterus cytoplasmic estrogen receptor shows a specifically displaceable and saturable binding to intact nuclei of mouse uterus after estradiol stimulation. The binding is complete after 3 hr at 0 degree C. The binding of the antibody correlates with the exchangeable estradiol binding activity of the nuclei over a 4-hr time course following in vivo injection of 17 beta-estradiol.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.