Identification of plasma membrane glycoproteins involved in the contact-dependent inhibition of growth of diploid human fibroblasts

Growth of normal, nontransformed cells is regulated by the interplay between growth stimulating compounds and growth inhibiting cell-cell contacts. We have previously shown that the growth of normal diploid human fibroblasts is mainly regulated by a specific class of plasma membrane glycoproteins (R. J. Wieser and F. Oesch (1986) J. Cell Biol. 103, 361-367). Because it was found that immobilization of the glycoproteins involved in contact-dependent inhibition of growth is an essential step in the recovery of the biological activity of the glycoproteins, we developed a technique for a first characterization of the active compounds. After SDS-PAGE separation of plasma membrane glycoproteins, they were transferred onto nitrocellulose. The nitrocellulose was cut along the separation track into circles which fit into wells of a 96-well microtiter plate. Culturing human diploid fibroblasts on the nitrocellulose circles resulted in characteristic growth patterns, which were dependent upon the source and the treatment of the plasma membrane proteins which had been separated. Five major inhibitory fractions with apparent molecular masses of 300, 170, 90, 50, and 25 kDa have been identified in plasma membranes from confluent fibroblast cultures.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

High frequency, heat treatment-induced inactivation of the phosphinothricin resistance gene in transgenic single cell suspension cultures of Medicago sativa.

Summary One descendant of the Medicago sativa Ra-3 transformant T304 was analysed with respect to the somatic stability of the synthetic phosphinothricin-N-acetyltransferase (pat) gene which was used as a selective marker and was under the control of the 5/3 expression signals of the cauliflower mosaic virus (CaMV) gene VI. In order to quantify gene instability, we developed a system for culturing and regenerating individual cells. Single cell suspension cultures derived from T304 and the ancestral non-transgenic M. sativa cultivar Ra-3, were established. The cells were regenerated into monoclonal calli. In transgenic calli, the phosphinothricin (Pt)-resistance phenotype was retained after more than 2 months of non-selective growth. In contrast, up to 12% of the suspension culture cells grown under non-selective conditions and at constant temperature (25° C) lost the herbicide-resistance phenotype within 150 days. Surprisingly, a heat treatment (37° C), lasting for 10 days, during the culture period resulted in an almost complete (95%) loss of the Pt resistance of the suspension culture cells. However, the frequency of cell division was identical in cultures grown under normal and heat treatment conditions. A biochemical test revealed that no phosphinothricin-N-acetyltransferase activity was present in heat treated, Pt-sensitive cells. The resistance level of the Pt-sensitive transgenic cells was equivalent to that of the wild-type cells. A PCR analysis confirmed the presence of the pat gene in heat treated, Pt-sensitive cells. From these results it is concluded that the Pt resistance gene was heat-inactivated at a high frequency in the M. sativa suspension cultures.     

Steroid-independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles in vitro

The content of prostaglandins of the E-group (PGE) or F-group (PGF) was determined by radioimmunoassay in rat ovaries and in homogenates of cultured Graafian follicles. Intraperitoneal administration of luteinizing hormone (NIH-LH-S18; 10 μg/rat) at 9.00 h on any day of the estrous cycle caused an increase in ovarian PGE content within 5 h. The response was greatest on the day of proestrus (940% rise), i.e. when the ovary contains large follicles, and least at metestrus (80%). Follicles explanted from proestrous rats before the preovulatory gonadotropin surge responded to addition of LH (1–5 μg/ml) to the culture medium with a 10 to 30-fold increase in PGE and a 5-fold increase in PGF accumulation over a 5-h-period. Follicle stimulating hormone (NIH-FSH-S9; 10 μg/ml) caused a similar rise in follicular PGE accumulation, even after treatment of the FSH preparation with excess of an antiserum to the β-subunit of LH. Stimulation of follicular PG accumulation was unimpaired during suppression of progesterone and estrogen synthesis by aminoglutethimide. It is concluded that these steroids play no part in the mediation of the LH-effect on follicular prostaglandin formation.     

Blockade of endotoxin-induced hypothermia by pretreatment with intracisternal 6-hydroxydopamine

Endotoxin-induced hypothermia was blocked in rats by pretreatment with intracisternally-applied 6-OHDA but not by systematic pretreatment with 6-OHDA or by pretreatment with the dopamine receptor blocking agent, pimozide. These data suggest that brain norepinephrine-containing neurons may in part mediate the thermic changes that result from challenge with endotoxin.     

The apparent absence of a pathway for synthesis of acetyl coenzyme A in pea chloroplasts: Lack of 14CO2 incorporation into lipids by the isolated organelle

Summary We have examined the extent to which isotopic lable derived from photosynthetically fixed ¹⁴CO₂ can be transferred to lipids by aqueously isolated chloroplasts of Pisum sativum. Although photosynthetically active, chloroplast preparations incubated with ¹⁴CO₂ showed little or no accumulation of label in lipids under any condition tested. Under identical conditions the chloroplasts were readily able to incorporate [¹⁴C]acetate into the lipid fraction; a fatty-acid synthesizing system was therefore operative in these chloroplasts.The essential failure of the isolated chloroplasts to incorporate label from fixed ¹⁴CO₂ into fatty acids supports the view that the organelle itself does not possess a self-contained pathway for the synthesis of acetyl coenzyme A, and favours the possibility that a shuttle mechanism involving the participation of extra-chloroplastic enzymes may be responsible for supplying the chloroplast with acetyl coenzyme A in vivo.     

Verhalten einer Gruppe von Brazzameerkatzen (Cercopithecus neglectus) im Heidelberger Zoo

For drawing phylogenetic conclusions we need a solid knowledge of those basic behavioural traits, which can be observed in many subgroups of primates and which do not seem to be influenced by environment. Details can be observed better in captivity than in the wild. Following the studies in behaviour of C. diana, for the first time the behaviour of C. neglectus was studied in detail during 2.5 months in the Zoo at Heidelberg. One group comprised an adult pair with 3 female offspring, one of them being taken out after ten days, and one half-year-old male infant. The second group comprised an adult male and a female, being brother and sister and children of the pair in the first group. An almost complete inventory of behavioural elements was collected, including comfort behaviour, intake of food, resting and sleeping, diurnal rhythm, postures in sitting, preference of places in the cage, ways of locomotion, play, vocalisation, reactions to disturbances, sexual behaviour, aggression and submission. The meaning of the individual elements was checked. The frequency of aggressive behaviour against the oldest children seems to be connected with an expulsion from the group. The extremely infrequent and in most cases merely intended sexual behaviour of the brother and sister-pair is discussed. It seems, together with the expulsion of the oldest children to be a means of avoiding incest.