Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Relative effects of cooling and warming rates on mammalian cells during the freeze-thaw cycle

The relative roles of cooling and warming rates on cell survival during a freeze-thaw cycle were investigated. Basically the faster the warming rate, the better the cells survive. One of the factors influencing this is the extended phase transition period at the slower thawing rates. The warming rate had a significant effect on cell damage and recovery, but this was not as great as comparative changes in the cooling rate were. This investigation also showed that under certain freeze-thaw conditions there was a lack of correlation between the two methods used for quantifying cell recovery (RI) and cell damage (PCR) as measured by radiochromate release. The analysis of the relationship between RI and PCR showed that PCR could be used to measure both lethal and nonlethal damage and enabled a clearer interpretation of cellular damage during cooling and thawing to be made.     

Studies on a possible pituitary effect of monoamines on luteinizing hormone release in ovariectomized rats

Incubation of anterior pituitaries from ovariectomized rats with LHRH and various concentrations of dopamine, norepinephrine or serotonin indicated that none of these neurotransmitters could decrease pituitary LH secretion in response to the releasing hormone. This indicated that the inhibitions of pulsatile LH release previously observed in our laboratory in ovariectomized rats in response to intraventricularly administered catecholamines or stimulation of brain serotoninergic neurons are due to central rather than pituitary effects of these transmitters.     

Age at puberty and estrous activity of straightbred and reciprocal crossbred gilts

Age at puberty, estrous activity and growth were evaluated in 294 straightbred and reciprocal crossbred gilts maintained in confinement conditions. Trial 1 used straightbred Landrace (L), Yorkshire (Y) and reciprocal crossbred (LY, YL) gilts. Trial 2 used LY and YL gilts, and Trial 3 used L and Duroc (D) reciprocal crossbred gilts (LD, DL). Daily observations for estrous activity with a mature boar were initiated at 150 days of age and continued until gilts reached 255 days of age. Gilts not exhibiting estrus by 255 days of age were examined by laparoscopy to determine reproductive status. In Trial 1, age at puberty was greater for Y gilts compared to L and reciprocal crossbred gilts. Differences between reciprocal crosses for age at puberty were not significant in any trial. In Trial 1, the percentage of gilts exhibiting regular estrous cycles at 195 days of age was lower for Y gilts than for the other three groups of gilts; however, by 225 days of age, this difference was not significant. In all trials, reciprocal crossbred gilts did not differ with respect to percentage exhibiting regular estrous cycles or in percentage prepubertal. In Trial 1, Y gilts had a higher percentage prepubertal at 255 days of age than the reciprocal crossbred gilts. The percentage of gilts exhibiting behavioral anestrus at 255 days of age did not differ among breed groups within any trial. Based on these observations, we concluded the breed maternal effects were small or nonexistent relative to direct additive and nonadditive genetic effects in the breed combinations that were investigated.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Changes in ultrastructures and enzyme activities during differentiation of myeloid leukemia cells to normal macrophages

Changes in ultrastructures and in enzyme activities were investigated electron microscopically, cytochemically and biochemically when mouse myeloid leukemia cells, Ml cell line, successfully differentiated to normal macrophages after incubation with a conditioned medium harvested from secondary embryo fibroblasts, or a lipopolysaccharide from Salmonella typhosa. The number of mitochondria increased significantly accompanied by the enhanced activity of cytochrome oxidase per cell, although the activity in each mitochondrion remained unchanged. The rough-surfaced endoplasmic reticulum elongated and often exhibited a concentrically multilayered lamellae. Glucose-6-phosphatase activity, a marker enzyme for the endoplasmic reticulum, also increased. Primary lysosomes were newly formed where acid phosphatase activity was positively demonstrated. Ten-nm cytoplasmic microfilaments, mainly forming bundles, and other microfilaments less than 6 nm wide were formed newly and abundant. Budding of type C viruses from the plasma membranes was reduced strikingly. Another established cell line, Mm-1, which spontaneously differentiated from the Ml cell line, was characterized completely by a macrophage, in which azurophilic granules (primary lysosomes), secondary lysosomes possessing strong activity of acid phosphatase and 10-nm microfilaments were most remarkable. These non-transplantable Mm-1 cells sometimes exhibited budding of viruses.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.     

Analysing the changing cell cycle

Details of the new methods of analysis of chain folding characterization of proteins proposed earlier (Srinivasan, Balasubramanian &; Rajan, 1975) and their application to six proteins are presented. Results pertaining to the angles θ and δ are presented.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

Cholecystokinin varies in the posterior pituitary and external median eminence of the rat according to factors affecting vasopressin and oxytocin

Using radioimmunoassay (RIA), the content of gastrin-cholecystokinin family peptide immunoreactivity (G-CCK-IR), in the posterointermediate lobe (PIL) of the rat pituitary, has been determined in several experimental conditions. G-CCK-IR levels are significantly higher in males than in females. Salt loading induces a significant decrease of G-CCK-IR in animals of either sex. In males, G-CCK-IR levels are lower than controls 21 days after either castration or daily subcutaneous oestradiol injections. Using immunocytochemistry, G-CCK-IR disappears from the external median eminence 21 days after adrenalectomy. Our results show that, in addition to sex difference, factors affecting the vasopressin and/or oxytocin levels in the posterior pituitary and external median eminence also affect G-CCK-IR in the same regions. Cholecystokinin may therefore be of importance in functions related to these hormones.     

DNA repair in UV-irradiated heteroploid cells at different phases of the cell cycle

The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.     

Reduction of anionic sites on the rabbit uterine surface during the preimplantation period

Studies have been carried out to analyze distribution of anionic sites on the uterine epithelium of the rabbit, using cationized ferritin as a label. A negatively charged glycocalyx was demonstrated by transmission electron microscopy on the luminal cell surface during estrus and days 5–7 of pregnancy. There was a general reduction of labeling from estrus and day 5 to 7 of pregnancy. At estrus and on day 5 and 6 of pregnancy, the results were similar on the meso- and antimesometrial sides of uterine horns and at or between egg recovery sites. At day 7, anionic sites were no longer detected antimesometrially facing the eggs. These results suggested that the progressive loss of anionic sites during the preimplantation period was due to the combined actions of uterus and egg and that this loss might play a role in blastocyst antimesometrial implantation.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

Synthesis of tubulin and actin during the preimplantation development of the mouse

The relative rates of synthesis of actin and tubulin during mouse preimplantation development have been investigated utilizing O'Farrell's two-dimensional polyacrylamide gel system and internal protein markers. During mouse preimplantation development, rates of protein synthesis remain low and are little changed until the 8-cell stage when a rapid increase is evident. From the 8-cell stage on, a much higher rate of synthesis is maintained. The rate of synthesis of actin remains also at a steady low level in the unfertilized and fertilized ovum. However, by the 8-cell stage actin synthesis has increased 10-fold. Our measurements include the blastocyst, at that point in development actin synthetic rates are almost 90-fold higher than in the unfertilized ovum. While this precipitous increase is proceeding, incorporation of [³H]leucine into total protein increases only 7-fold. Synthesis of actin in the blastocyst represents 5.7% of total protein synthesis. The rate of tubulin synthesis, unlike actin, more closely parallels the increments in total protein synthetic rates. At the blastocyst stage it has increased 14-fold and its synthesis represents almost 2% of total protein synthesis. These results are discussed with reference to some of the physiological changes taking place during preimplantation development.     

Procoagulant effect of phenobarbital in rats

Daily administration of phenobarbital, 75 mg/kg ip for 4 days, to adult male Sprague-Dawley rats produced a pronounced increase of prothrombin complex activity (PCA) in plasma, i.e. a decrease of the prothrombin time. This effect persisted for 4 to 5 days after the last dose of phenobarbital. The rate constant for the decline of PCA after administration of a PCA synthesis-blocking dose of warfarin was not affected by treatment with phenobarbital but the rate of synthesis of PCA was increased appreciably. Thus, the phenobarbital-induced increase of PCA was caused by increased synthesis of one or more clotting factors.     

Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock

The cholesterol content of spermatozoa with highly unsaturated phospholipids, i.e., ram and bull, which are very susceptible to cold-shock was compared with that of rabbit and human spermatozoa which have more saturated phospholipids and a greater resistance to cold-shock. The level of cholesterol in the seminal plasma was also estimated.Cholesterol was present in ram and bull spermatozoa in comparable amounts (280–346 μg) and at approximately half the level (in micrograms per 10⁹ spermatozoa) in rabbit and human spermatozoa (545–556 μg). The molar ratio of cholesterol: phospholipid in rabbit and human spermatozoa was 0.88 and 0.99, respectively, which is similar to the ratio of these lipids in erythrocytes and higher than the ratio of 0.38 and 0.45 for ram and bull spermatozoa, respectively. This ratio is of some importance in determining the nature and degree of packing in the spermatozoan membrane; higher levels of cholesterol result in a more cohesive, rigid, and impermeable structure. A definite relationship is apparent between the ratio of cholesterol: phospholipid, the ratio of polyunsaturated: saturated phospholipid-bound fatty acids, and the susceptibility of the spermatozoa to cold-shock.     

Age-related alterations in plasma membrane glycoprotein content and scheduled or unscheduled DNA synthesis.

WI-38 cells of various ages and SV40-transformed WI-38 cells were examined for differences in plasma membrane composition of glycoproteins and DNA synthesis. Sialic acid per milligram of protein content of the membranes of WI-38 cells decreased with passage of time in culture. Other glycoprotein fractions and alkaline phosphatase activity disappeared in the WI-38 cells with passage of time in culture (Phase III). Studies of DNA repair correlated with changes observed in the plasma membrane glycoprotein content of WI-38 cells over a passage of time in culture were also reported. Both the extent and rate of ultraviolet-induced unscheduled DNA synthesis remained relatively constant during the passage of the WI-38 cells until late phase III. At that time the extent of unscheduled DNA synthesis was measurably reduced. The number of cells in a population of phase III cells able to perform semiconservative DNA synthesis diminished with age in culture but not to an extent capable of explaining the observed changes seen in membrane composition of semiconservative DNA synthesis during passage of the cells in culture. Cells with an extended lifespan SV40-transformed WI-38 (VA 13.2 RA) cells, did not vary in membrane composition, semiconservative DNA synthesis, or unscheduled DNA synthesis over 200 serial subpassages of the cells in culture.