Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.     

Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomnnose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B withA. saitoi (1–2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.     

Isolation and mapping of polymorphic cosmid clones used for sublocalization of the multiple endocrine neoplasia type 1 (MEN1) locus

Summary Multiple endocrine neoplasia type 1 (MEN1) is characterized by neoplasia of the parathyroids, the pancreas, and the pituitary. Tumorigenesis involves unmasking of a recessive mutation at the MEN1 locus, which has been mapped to the centromeric part of chromosomal region 11q. In order to localize the MEN1 gene further and to make its isolation possible, a number of new markers were isolated. Two radiation-reduced somatic cell hybrids were identified that only contained markers close to and flanking the MEN1 region. DNA from these hybrids was used for the construction of a cosmid library, and clones containing human inserts were isolated. In addition, cosmid clones were isolated for locus expansion of 7 other markers that were mapped to the 11q12–13.2 region. The 33 newly isolated clones together with 25 previously published markers from this region were analyzed in a panel of radiation-reduced somatic cell hybrids. From the hybridization pattern, the region was divided into 11 parts. New restriction fragment length polymorphisms were identified in 7 of the newly isolated cosmid clones and in one plasmid. These were then used to sublocalize meiotic cross-overs more precisely in two MEN1 families, thus refining the mapping of the disease gene.     

Effect of pyridoxine on tumor necrosis factor activitiesin vitro

Clinical trials with tumor necrosis factor (TNF) as an antitumor agent have so far given rather disappointing results. In this study we show that the naturally occuring vitamin B₆ compound, pyridoxine, enhances TNF-induced cytolysis of three subclones of a mouse fibrosarcoma cell line (WEHI 164). The degree of pyridoxine-induced enhancement of TNF cytotoxicity seems to be dependent on the cells sensitivity to TNF, as the enhancement was much more pronounced in the relatively TNF resistant subclone act-R(cl.12)-WEHI 164, than in the very TNF sensitive subclone WEHI 164 clone 13. Furthermore, our study shows that pyridoxine, in contrast to its enhancing effect on TNF-induced cytotoxicity, rather inhibits TNF-induced growth of human FS-4 fibroblasts. Pyridoxine also enhances lymphotoxin (LT)-induced tumor cell killing and inhibits LT-induced fibroblast growth. Pyridoxine is a relatively non-toxic agentin vivo. Our results suggest that a combination of TNF and pyridoxine may be more efficient than TNF alone, in the treatment of cancer patients.     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

abetd-Xylose metabolism in Aureobasidium pullulans: effects of aeration and vitamins

Summary The yeast-like organism Aureobasidium pullulans efficiently converted abetd-xylose to cell mass (Y _X/S=0.45 g·g^–1) with negligible production of polyols (Y _P/S=0.003 g·g^–1) under aerobic conditions. A. pullulans grown semiaerobically exhibited different fermentation capacities in seven basal (vitaminless) medium and medium containing a mixture of seven vitamins. It was found that under semiaerobic conditions a mixture of vitamins significantly enhanced production of ethanol from abetd-xylose, resulting in a 15-fold higher yield coefficient of ethanol (Y _E/S=0.22 g·g^–1) as compared to that achieved in vitaminless medium. This increase in ethanol production was accomplished at the expense of cell mass. A. pullulans produced extremely low amounts of polyols throughout all aerobic and semiaerobic experiments. A. pullulans displayed strictly NADPH-linked xylose reductase and NAD⁺-linked xylitol dehydrogenase activities.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.