Chromatin- and nuclei-directed ribonucleic Acid synthesis in sugar beet root

The synthesis of RNA by chromatin-bound RNA polymerase prepared from sugar beet (Beta vulgaris) root tissue is completely dependent on the presence of a divalent metal (Mg²⁺ or Mn²⁺) and the presence of four ribonucleoside triphosphates. Accumulation of labeled acid-insoluble product is inhibited by the addition of RNase and actinomycin D to the reaction. When beet root slices are washed for 25 hours, chromatin-associated RNA polymerase activity increases 7-fold over that of unwashed tissue. This enzyme activity declines with further washing. DNA template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, was also found to follow a pattern similar to that for RNA polymerase. Nearest neighbor frequencies of the RNA synthesized by chromatin isolated from unwashed and washed tissue are different.     

Nitrogen fixation associated with roots of Kallar grass (Leptochloa fusca L. Kunth)

Summary An ecological survey was made to measure the N₂-fixing activity in the rhizosphere of a salt-tolerant grass,Leptochloa fusca (L.) Kunth. Samples were obtained every month at two sites over a period of one year. Soil cores, unwashed, washed and surface-sterilized roots were subjected to acetylene reduction assay (ARA). ARA values up to 50 nmoles h^–1 for soil cores, 1095 nmoles g dry root^–1 h^–1 for unwashed roots, 4929 nmoles g dry root^–1 h^–1 for washed roots and 2494 nmoles g dry root^–1 h^–1 for surface-sterilized roots were observed but for most samples the range was 1–200 nmoles g dry root^–1 h^–1. A lag period of 5–7 h was observed before the onset of N₂-fixing activity by excised roots and O₂ levels had no effect on this lag. Values for roots incubated without preincubation were similar to those for unwashed preincubated roots. Activity was highest in September, October and November when the temperature is not very high and photosynthetic activity is reasonably good. N₂-fixing-bacteria were counted on the same samples by plate count and MPN methods, the latter being estimated on the basis of ARA and pellicle formation. Fairly high numbers of bacteria (10⁴–10⁷) were recorded in the histoplane fraction which indicates the presence of diazotrophs in the inner cells of grass roots.     

The Presence of a Ribonuclease of High Molecular Weight in Sugar Beet Storage Tissue

Soluble ribonuckasie activity in sliced root tissue or sugar beet (Beta vulgaris) decreased to 30% of initial levels during the first 15 hours of aeration in sterile phosphate buffer. Activity increased with continued aeration reaching a maximum at 30 hours (85% of initial levels). Ribonuclease activity was isolated from unaerated tissue and partially purified by precipitation with acid and ammonium sulfate and by Sephadex chromatography. Enzyme activity was linear with respect to enzyme and substrate concentrations. The enzyme exhibited a substrate preference for RNA with no activity in the presence of native or denatured DNA. Elution of the enzyme from a Sephadex G-150 column indicated a molecular weight of 155,000. This uniquely large ribonuclease had no phosphodiesterase activity, was unaffected by ethylenediamine tetraacetate, and was inhibited by increasing Nig²⁺ concentrations.     

Loss of cell components during rehydration of dried Rhodotorula glutinis and its implications for lead uptake

Microbial cells are routinely dried and ground before they are used in metal biosorption studies. In this work, a metal biosorbent was prepared by drying biomass of the yeast Rhodotorula glutinis in an oven at 70°C for 24 h followed by grinding. Two forms of the prepared biosorbent particles, washed and unwashed, were examined for their ability to remove lead from solution. It was found that the unwashed biosorbent exhibited higher lead uptake than the washed biosorbent. Analysis of the supernatant of washed cells incubated in water and that of unwashed cells incubated in lead solution revealed the presence of protein, carbohydrates, organic acids and inorganic phosphate. Overall, the washed and unwashed cells leached, respectively, 14.5 and 13.4% of their initial dry weight (100 mg). Acid‐base titration data revealed that the leached components contained several potential binding sites for metal cations with carboxyl and phosphoryl groups being particularly important. The higher level of lead uptake exhibited by the unwashed biomass was attributed to the fact that it leached smaller amounts of cell constituents with proton binding sites relative to the washed cells.     

Benzodiazepine Receptor Binding Following Amygdala-Kindled Convulsions: Differing Results in Washed and Unwashed Brain Membranes

The binding of [3H]flunitrazepam and [3H]RO5-4864 was measured in unwashed brain homogenates and in extensively washed brain membranes from amygdala-kindled and "yoked" control rats sacrificed 2 weeks following the sixth stage 5 convulsion. In unwashed homogenates, [3H]flunitrazepam binding was reduced in both the hypothalamus and ipsilateral right cortex of kindled rats (unchanged in other areas). In washed brain membranes, [3H]flunitrazepam binding was unaltered in these regions; it was bilaterally elevated, however, in both the amygdala and hippocampus (unchanged in other areas). In washed membranes, the in vitro addition of gamma-aminobutyric acid enhanced [3H]flunitrazepam binding to a similar extent in kindled and control membranes. These data indicate that the type of benzodiazepine binding abnormality observed after kindling depends on the type of tissue preparation employed in the assay procedure.     

Effects of a partially purified factor from chick embryos on macromolecular synthesis of embryonic pancreatic epithelia

Essentially normal development of early embryonic pancreatic epithelium occurs only in the presence of mesenchymal tissues (Golosow and Grobstein, 1962), or a particulate fraction (MF) obtained from extracts of chicken embryos (Rutter et al., 1964). We have shown that this fraction also stimulates the incorporation of thymidine-³H into DNA. This stimulatory activity was detected in particulate fractions from homogenates of several mesodermal tissues from rat and chick embryos, as well as in fibroblasts cultured from these tissues, but not in embryonic epithelial tissues. This activity may thus be related to the mesodermal tissue requirement for pancreatic development. MF was solubilized and partially purified from homogenates of chick embryos. It is stable to collagenase, hyaluronidase, and neuraminidase. Activity is lost by heating and by treatment with trypsin. It is presumed, therefore, that the factor is associated with a protein that is not collagen.The effects of the MF upon macromolecular synthesis were tested in pancreatic tissues from 12-day rat embryos. When isolated epithelia were cultured in the absence of mesoderm or MF, the rate of thymidine-³H incorporation into DNA decreased to low levels. The specific activities of DNA polymerase and deoxycytidylate deaminase in epithelial extracts also declined. In contrast, the rate of thymidine-³H incorporation into DNA increased 5- to 8-fold over the initial rates in epithelia cultured with MF. Concurrently DNA polymerase activity in tissue extracts increased by 2- to 3-fold; deoxycytidylate deaminase activity declined slightly.MF also affected RNA and protein synthesis. The rate of leucine-³H incorporation into protein and uridine-¹⁴C incorporation into RNA in isolated pancreatic epithelia was comparable to that of intact rudiments. Cultures in the presence of MF increased these rates severalfold after 20 hr. These results suggest that MF, and by implication, mesoderm, may supply a growth factor for epithelial tissue and thus serves a permissive rather than a determining role in the differentiation process in pancreatic development.     

The effects of a settled industrial domestic sewage works effluent from percolating filters on the embryo viability and hatching success of rainbow trout, Salmo gairdneri Richardson

The mortalities of washed and unwashed rainbow trout embryos exposed to Birmingham tap water, 50% and 100% settled effluent from filter beds has been compared. Differences in the hatching success between washed and unwashed embryos in the three treatments were also compared and the effect of saprophytic microbial growths assessed. There was a significant difference in mortality between washed embryos exposed to tap water (5%) and 50% effluent (12%) and 100% effluent (12%) when dissolved oxygen levels were maintained near 100% air saturation value. Mortalities of unwashed embryos were 4%, 26%, and 26% respectively for the three treatments. Hatching success of washed embryos was similar in the three treatments but unwashed embryos were significantly affected by the effluent treatments because of Sphaerotilus growths.     

Acrocalymma medicaginis and Phomopsis sp. as Causal Agents of Crown Rot of Lucerne in Australia

Fungi associated with crown rotting were isolated from 3 year-old lucerne plants growing in sandy and loam soils in an irrigated stand, declining in productivity, at Langhorne Creek, South Australia. The method of isolation markedly affected the range of species recovered. Fusarium spp. and Phomopsis sp. were isolated from washed and surface sterilized crowns, whereas a larger range of fungi, especially Acrocalymma medicaginis, was isolated from unwashed crowns. A. medicaginis was most frequently isolated from rotted tissue flecked with red, while a species of Phomopsis was the dominant fungus in whitish tissue containing zone lines, especially in crown branches. A. medicaginis and Phomopsis sp. caused crown rotting of mature, wounded lucerne plants at temperatures equivalent to summer conditions at Langhorne Creek. There was no evidence that Fusarium oxysporum or F. solani, two of the most frequently isolated species, were pathogens of mature lucerne plants.     

Effects of colloidal fouling and gas sparging on microfiltration of yeast suspension

Cross-flow microfiltration is an important step in separating Baker’s yeast (Saccharomyces cerevisiae) from aqueous suspension in many processes. However the permeate flux often declines rapidly due to colloidal fouling of membranes and concentration polarisation. The present work explores the possibility of maintaining acceptable permeate flux by co-current sparging of gas along with the feed, which would scour away colloidal deposits and reduce concentration polarisation of membranes. In this work, both washed and unwashed yeast were used to study the effect of washing to reduce protein fouling of membranes. It was found that permeate flux increased by 45% for liquid throughput of 75 kg/h for a feed concentration of 2.0 kg/m³ of washed yeast as compared with unwashed yeast suspension without gas sparging. For washed yeast suspension, the increase in gas flow rate from 0.5 lpm to 1.5 lpm (30 l/h to 90 l/h) had beneficial effect on permeate flux. It is concluded that in the present case, the gas flow rate should be less than or equal to the liquid flow rate for enhancement of permeates flux.     

Fructose 2,6-bisphosphate in human erythrocytes

The fructose 2,6-bisphosphate concentrations in unwashed, washed, and leukocyte-free erythrocytes were compared. The concentration in washed red cells was 31 +/- 15 pmol per ml of cells (mean +/- S.D., n = 6). The concentration in unwashed erythrocytes was at least twofold higher, but the value in washed red cells was not due to leukocyte contamination because it did not decrease further when washed cells were passed through an Imgard column, which would have removed any remaining leukocytes. No platelets were detected among the washed erythrocytes. Thus, the concentration in erythrocytes after washing was ascribed solely to these cells. The fructose 2,6-bisphosphate concentration did not change when the glycolytic activity varied with pH, indicating that this compound is not involved in the regulation of carbohydrate metabolism in erythrocytes under these conditions.     

On the difference between washing-induced and fusicoccin-induced K+ uptake in maize root segments

A comparison between fusicoccin (FC)-induced and washing-induced increase of K⁺ uptake in maize root segments is reported.K⁺ uptake rate nearly doubles after 4 h of washing as compared to the rate in the unwashed tissue. The stimulation of K⁺ uptake by FC is the same (approx. 1.6 μmol × g fresh wt.⁻¹ × 30 min⁻¹) both in unwashed and in washed tissue.Washing-induced increase of K⁺ uptake is strongly inhibited if 10⁻² M KCl is present in the washing solution; on the contrary, FC-induced increase of K⁺ uptake seems independent of the presence of KCl in the medium during the washing period.The inhibition of protein synthesis completely prevents the stimulation of K⁺ uptake induced by washing, while it only partially inhibits the effect of FC on the same process.These results, taken as a whole, suggest that the activation of K⁺ uptake induced by washing and that induced by FC depend on two different mechanisms.     

Embryos produced from fertilization with bovine viral diarrhea virus (BVDV)-infected semen and the risk of disease transmission to embryo transfer (ET) recipients and offspring

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems in cattle. For that reason, several countries have eradicated the disease, and some others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for artificial insemination (AI), there is no evidence to indicate whether the resulting embryos, when used for embryo transfer, can lead to the transmission of BVDV to recipients or offspring. For this experiment, semen from a bull persistently infected with BVDV (10⁵ 50% tissue culture infective doses/mL NY strain) was used for insemination (two times at estrus) of BVDV-seronegative, superovulated cows (N = 35). Embryos were collected 7 days after insemination and subsequently were washed according to the International Embryo Transfer Society recommendations or left unwashed. Out of 302 collected oocytes and embryos, 173 (57%) were fertilized and the remaining 129 (43%) had degenerated. Infectious BVDV was detected in 24% (17/71) of unwashed and 10% (8/77) of washed embryos, and in all (N = 11) follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. After transfer of 39 washed embryos to 27 BVDV-seronegative recipients, 12 (44%) cows became pregnant and 17 calves free of BVDV and BVDV antibodies, including five sets of twins, were born. After embryo transfer, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In conclusion, results herein suggest that BVDV can be transmitted by AI resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to International Embryo Transfer Society and the World Organization for Animal Health guidelines do not cause BVDV transmission to recipients or their offspring.     

Effect of corticosterone on epididymal lipids in pubertal rat

Serum corticosterone excess was induced by the administration of corticosterone acetate to adrenal intact rats. Different lipid classes were studied in unwashed and washed (epididymal sperm and fluid free) caput and cauda epididymides. The unwashed caput epididymidis registered a significant decrease in total lipid, cholesterol and phospholipid while total glyceride glycerol and its fractions were not altered after corticosterone treatment. Among phospholipid fractions phosphatidyl inositol, choline and ethanolamine showed a significant decrease. Unlike the unwashed caput epididymidis, the washed caput region recorded a marked increase in total lipid, glyceride glycerol and its fractions. However, total lipid in the washed cauda region significantly increased and the increase was mainly due to triacyl glycerol. Though the phospholipid fractions phosphatidyl choline and ethanolamine showed an increase, the total phospholipid was not altered significantly. Serum testosterone and prolactin registered a significant decrease while gonadotropins were unaltered. On the withdrawal of corticosterone treatment, all the lipid classes turned to normalcy along with serum testosterone and prolactin. It is concluded that corticosterone excess favours lipid accumulation in the sperm free epididymal tissue and its influence on epididymis is region specific and reversible.     

Reconstitution of cyanobacterial photophosphorylation by a latent Ca2+-ATPase.

Photosynthetic membranes derived from sonic extracts of the cyanobacterium $\text{Spirulina}\text{platensis}$ contain a latent Ca⁺²-ATPase which is activated by exposure to trypsin. When sonic membranes are washed with ethylenediaminetetraacetic acid, the ATPase is removed from these membranes with an accompanying loss of photophosphorylation activity. The latent ATPase activity solubilized by washing has been partially purified, and addition of the enzyme to depleted membranes restores photophosphorylation activity to levels approaching 50% of the rates observed in unwashed membranes. These data indicate that this ATPase is the coupling factor responsible for photosynthetic energy transduction in $\text{Spirulina}\text{platensis}$.     

The Significance of Exometabolites in the Formation and Operation of the Soybean–Rhizobium Symbiosis

The effect of various inoculates of the soybean-specific strain of nodule bacteria Bradyrhizobium japonicum 634b (unwashed cells, cells washed from the exopolysaccharide–protein complex, and cells combined with the complex) on the formation and operation of soybean–rhizobium symbiosis. It was shown that addition of the exopolysaccharide–protein complex doubled the ability of the microsymbiont to form nodules, nodule weight, and the nitrogenase activity of the nodules. Bradyrhizobium japonicum 634b cells washed from exometabolites had lower indices of symbiotic activity than their intact counterparts.     

Localization of fatty acid hydroperoxide cleavage activity in membranes of cucumber fruit

The subcellular localization of the fatty acid hydroperoxide cleavage enzyme from cucumber fruit has been studied. Activity from the flesh tissue has been located in 3 fractions; plasma and Golgi membranes and endoplasmic reticulum, at equilibrium densities on sucrose gradients of 1.17, 1.15 and 1.12 g/cm³ respectively. Enzymatic activity and electron microscopy studies were carried out to identify plasma and Golgi membranes. Little activity was associated with microbodies (1.23), plastids (1.21) and mitochondria (1:19 g/cm³). However, chloroplasts isolated from the peel of the cucumber fruit contained a large amount of hydroperoxide cleavage activity.     

SILICON METABOLISM IN DIATOMS. VI. SILICIC ACID UPTAKE BY A COLORLESS MARINE DIATOM,NITZSCHIA ALBA LEWIN AND LEWIN12

Cells of the colorless, heterotrophic diatom Nitzschia alba, after removal from the culture medium, were able to absorb silicic acid from a salt solution lacking carbon, nitrogen, and phosphorus. Silicic acid uptake continued for approximately 12 hr. At low cell densities (ca. 2.5×10⁵ cells/ml), the cell number doubled under these conditions. At high cell densities (ca. 2 ×10⁶ cells/ml) no cell division resulted. When such cells were washed with a salt solution, their ability to absorb silicic acid was somewhat impaired. The degree of impairment became progressively more pronounced after subsequent washing treatments. A heat-stable factor washed from the cells and present in the first “wash water” was able to restore completely the ability of washed cells to absorb silicic acid. The factor was not identified. Aspartic acid (5 ± 10^-4 M) or glutamine (5 ±10^-4 M) when added to the saline solution similarly promoted complete recovery. A t such concentrations, these substances had only a slight effect on unwashed cells. A solution of 5 ±10^-4 M Na glutamate (or aspartic acid plus glutamine) had an men more pronounced effect, and in addition promoted cell division or growth" of unwashed cells. Several other amino acids and other compounds tested were apparently without effect.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

The Electrophoretic Velocity of Human Red Cells, of Their Ghosts and Mechanically Produced Fragments, and of Certain Lipid Complexes

Ghosts prepared in CO₂-saturated water from unwashed human red cells can be fragmented mechanically, but ghosts from thrice washed cells cannot. If the ghosts are prepared by freezing and thawing, this difference is not observed. The electrophoretic velocity varies also with the way in which the ghosts are prepared. The pH-mobility dependence of washed red cells flatten off to a plateau at pH 9, and the electrophoretic velocity is zero at about pH 2. Ghosts prepared by freezing and thawing have almost the same pH-mobility dependence, but if the ghosts are prepared in CO₂-saturated hyptonic saline, the mobility at pH 9.4 is 0.75 times that of washed cells. Fragments of ghosts of unwashed red cells have a smaller mobility than that of the red cells. Trypsin reduces the mobility of washed red cells and of ghosts. Sols of lipid complexes (lecithin, cephalin, and lipositol), at varying pH's, have a mobility 1.2 times that of the washed red cell. The pH-mobility relation is otherwise similar. These complexes can be coated with dextran and trypsin.     

The significance of exometabolites in the formation and operation of the soybean-rhizobium symbiosis

The effect of various inoculates of the soybean-specific strain of nodule bacteria Bradyrhizobium japonicum 634b (unwashed cells, cells washed from the exopolysaccharide-protein complex, and cells combined with the complex) on the formation and operation of soybean-rhizobium symbiosis. It was shown that addition of the exopolysaccharide-protein complex doubled the ability of the microsymbiont to form nodules, nodule weight, and the nitrogenase activity of the nodules. Bradyrhizobium japonicum 634b cells washed from exometabolites had lower indices of symbiotic activity than their intact counterparts.