Amplified Fragment Length Polymorphisms (AFLPs) detected with non-radioactive digoxigenine labelled primers in three plant species

A protocol for the detection of AFLPs with the non radioactive digoxigenine labelling is presented. The protocol has been tested on DNA samples from three different plant species. The AFLP technique was used for the first time in these species. The sensitivity and reliability of the digoxigenine labelled primers in the AFLP technique was of the same order as the sensitivity and reliability of the radioactive assay. No major adjustments of the current standard AFLP protocols is necessary to use the digoxigenine labelled primers.     

Stocking success of newly hatched pike evaluated by radioactive strontium (85Sr) marking

The stocking of newly hatched pike Esox lucius labelled with radioactive strontium (⁸⁵Sr) and later electrofishing of the same area was found to be a workable tool in assessing the success of pike stockings. ⁸⁵Sr‐labelled individuals constituted 96% of the 0+year pike catch from late summer electrofishing in the stocked littoral areas of the heavily regulated Lokka Reservoir. The density of 0+year pike reached 2·1 individuals 100 m⁻² in the stocked areas in comparison to 0·3 individuals 100 m⁻² in the reference areas. In the three other stocked lakes the proportion of labelled pike in the catch ranged from 0 to 42% in late summer. The results suggest that pike stockings are profitable in filling empty or nearly empty 0+year pike habitats of regulated lakes or other waterbodies, where the natural reproduction of pike has declined due to human activities.     

The frequency and distribution of isolabelling in Chinese hamster chromosomes after exposure to x-rays

2 experiments were carried out, following the same scheme. Chinese hamster cells (line CHEF-125) were cultivated for 4 h in the presence of tritiated thymidine ([³H]TdR) (Expt. 1, 0.5 μCi/ml; Expt. 2, 0.1 μCi/ml). After removal of the isotope, some of the cultures were X-irradiated in stage S-G₂ preceding M₁ (series S-G₂ I), others in stage S-G₂ preceding M₂ (series S-G₂ II).Analysis of the colchicine-C-anaphase of M₂ cells showed that in both experiments the X-rays produced an increase in the isolabelling regions only if given in the S-G₂ stage preceding M₁. On the other hand, the X-rays produced an increase in sister chromatid exchanges (SCEs) in both series of the second experiment. In the first experiment, where the controls had a high frequency of SCEs, no increase in SCEs was recorded in either of the two series treated with X-rays. The frequency of the isolabelling regions, in relation to the total labelling (Expt. 2), was 1.4% in the control series, 2.5% in series S-G₂ I and 1.3% in series S-G₂ II.Analysis of the distribution of the isolabelled regions along the chromosome showed that the regions furthest from the centromere were those most involved in this phenomenon and that, for the most part, the isolabelling was not directly associated with an exchange.The results have been interpreted, in accordance with the theory put forward in a previous work, to mean that SCEs and isolabelling regions are due to different phenomena, the former resulting from an exchange involving the whole chromatid, the latter from subchromatid exchanges probably involving a single polynucleotide strand.     

Effect of adenosine 3',5'-cyclic monophosphate on the RNA and DNA synthesis and cell proliferation of rat hepatocytes in primary culture: a radioautographic study.

The effect of a 24-h treatment with various doses (from 1.5-10-minus 8 to 3.0-10-minus 3 M) of adenosine 3',5'-cyclic monophospahte (cAMP) on morphometric parameters, [5--3H]uridine radioactivity concentration (URC), [methyl--3H]thymidine [Me--3H]-Tr) labelling index per hour (L.I./h) and per cent mitotic index (M.I.%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographic methods. In such cells cAMP was found to induce: (1) a reduction of the apparent surface area (ASA) of total nucleoli, karyoplasm and cytoplasm; (2) significant increases in URC of all the subcellular compartments at all the dosages employed (only cAMP at 1.5-10-minus 8 M did not change karyoplasmic and cytoplasmic URC values); (3) marked increments in [Me--3H]Tdr L.I./h and M.I.% from the lowest dose up to 1.5-10-minus 4 M; at higher doses the L.I./h and M.I.% were less stimulated or approached control values. In cultured rat hepatocytes, adenosine-5'-phosphate (5'-AMP) (1.5-10-minus 4 M per 24 h) increased the karyoplasmic and total cell ASA, the lone total nucleolar URC and both the L.I./h and M.I.%. However, these metabolic effects were significantly less intense than those elicited by isomolar cAMP. Theophylline (Theo) (5.5-10-minus 5 M per 24 h) reduced the in vitro rat hepatocyte total nucleolar ASA but affected neither other morphometric nor any of the URC values. The same dose of Theo plus cAMP (1.5-10-minus M) had no morphometric effect but significantly increased the URC values of all primary rat hepatocyte compartments. Actinomycin D (DAct) (0.1 mug/ml per 24 h) plus cAMP (1.5-10-minus 4 M) decreased the cultured rat hepatocyte total nucleolar ASA but enlarged that of karyoplasm and cytoplasm and, further, markedly curtailed all the compartmental URC values. These data support the hypothesis that cAMP amplified the template activity of the liver chromatin and accelerates the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.     

Peroxisomes of soybean (Glycine max) root nodule vascular parenchyma cells contain a "nodule—specific" urate oxidase

The cortex of soybean ( Glycine max L. cv. Centennial) nodules contain an organellerich layer of vascular parenchyma tissue, which encircles the elaborate vascular tissue of the nodule. Peroxisomes with small, electron-opaque nucleoids are found in the vascular parenchyma cells. Positive cytochemical staining for catalase (EC 1.11.1.6) confirms their morphological identification as peroxisomes. Activities of both glycolate oxidase (EC 1.1.3.1) and urate oxidase (EC 1.7.3.3) were detected cytochemically in these peroxisomes. Nodule-specific urate oxidase was localized principally in the nucleoid region of these vascular parenchyma peroxisomes, as indicated by immunogold labelling using antibodies against nodulin-35, the nodule-specific urate oxidase. The density of urate oxidase immunogold labelling in the vascular parenchyma peroxisome nucleoid is similar to that of the more well-characterized interstitial cell peroxisomes of the infected zone. These results show that the induction of nodule-specific urate oxidase may be induced in tissue outside of the infected zone.     

Inactive Monomeric Acetyleholinesterase in the Low-Salt-Soluble Extract of the Electric Organ from Torpedo marmorata

Proteolytic fragmentation of [3H]diisopropylfluorophosphate-labelled catalytic subunits of different molecular forms of acetylcholinesterase demonstrates that all forms extracted from the electric organ from Torpedo marmorata are true acetylcholinesterases. This is supported by immunochemical results showing that the radiolabelled polypeptides are readily recognized by specific anti-acetylcholinesterase antibodies. Although distinct structural differences exist, all forms contain a similar peptide carrying the serine hydroxyl of the esteratic subsite. Dimeric, detergent-soluble acetylcholinesterase is present in the low-salt-soluble extract (Mr of the catalytic subunit 66,000) together with a monomeric form (apparent Mr 76,000). This monomeric polypeptide is hydrophilic, enzymatically inactive, and might represent a precursor of the asymmetric forms of acetylcholinesterase.     

Specific binding of three neurotoxins with phospholipase A2 activity to synaptosomal membrane preparations from the guinea pig brain

Snake presynaptic toxins such as crotoxin, -bungarotoxin and taipoxin block neuromuscular transmission through inhibiting the release of acetylcholine by their phospholipase A₂ activities. On the other hand, many other phospholipase A₂s show little neurotoxicity. It is likely that the difference lies in whether high affinity binding to nerve cell membranes exists or not. To test this idea, crotoxin, -bungarotoxin and taipoxin were first radioactively labeled with Na(¹²⁵I) without loss of their neurotoxicity. Using the radioactive toxins we have found that each of the three showed specific binding to synaptosomal membranes from guinea pig brain. In contrast, we could not detect specific binding of a non-neurotoxic pancreatic phospholipase A₂. Crotoxin and taipoxin, but not -bungarotoxin, also bound specifically to membrane preparation from other tissues. The binding of each toxin was not greatly affected by the other two toxins. The photoaffinity labeling technique has been used to obtain further information about the components which bind crotoxin. For this purpose, (¹²⁵I) crotoxin was derivatized with N-hydroxysuccinimidyl-4-azidobenzoate. Autoradiographic analysis of the membranes following photoirradiation in the presence of the modified crotoxin revealed that an 85K dalton component was preferentially covalently conjugated with the crotoxin analogue in a specific manner.On leave from Department of Biochemistry and Biophysics, University of Hawaii, School of Medicine, Honolulu, Hawaii.     

Nitrogen metabolism in cultures of Tabernaemontana divaricata

A cell suspension culture from Tabernaemontana divaricata was fed with ¹⁵N-labelled ammonium or nitrate. The incorporation of label in free amino acids, protein amino acids and indole alkaloids was determined. Ammonium was found to be used more extensively than nitrate in the biosynthesis of these compounds. For tryptamine considerably lower labelling percentages were found than for the indole alkaloid O-acetylvallesamine and the amino acids. This indicates a vacuolar pool of tryptamine, formed at the beginning of the culture-period and not available for further alkaloid biosynthesis.     

Cell cycle dependency of monoclonal antibody production in asynchronous serum-free hybridoma cultures

The cell cycle kinetics of F3(B6) mouse hybridoma was examined by immunocytochemical staining of bromodeoxyuridine incorporated into the DNA of exponentially growing cells in three different cultures: one supplemented with 10% fetal bovine serum and two adapted to serum-free media, TABIES and BITES. The serum-free cultures, particularly the BITES, had longer cycling times and higher specific antibody production rate. Both observations were correlated to the prolongation of the G₁ phase traverse time and substantiated with a starvation blocking experiment.