Evidence for functional hemizygosity at the Emtr locus in CHO cells through segregation analysis

The hypothesis of functional hemizygosity at the emetine-resistant (Emt^r, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emt^r and Thg^r (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emt^r-Thg^r CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thg^r segregants were obtained at similar frequencies (10^?2–10^?3) from all the hybrids. The frequency of segregation of the Emt^r marker, however, was similar to that of Thg^r only in the CHO × CHO hybrids and was much lower (10^?4–10^?6) than the CHO × other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emt^r is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Inactivation of Glutamine Synthetase by Ammonia Shock in the Gram-Positive Bacterium Streptomyces cattleya

In cultures of the gram-positive bacterium Streptomyces cattleya, a rapid inactivation of glutamine synthetase was seen after ammonia shock. pH activity curves for ammonia-shocked and control cultures are shown. A peak of glutamine synthetase activity was seen during fermentation for production of the antibiotic thienamycin.     

Influence of dicarboxylic phosphatidylcholines on phosphatidylcholine liposomes as revealed by gel chromatography and electron microscopy

The effect of dicarboxylic phosphatidylcholines (glutarylphosphatidylcholine) on the structural changes of phosphatidylcholine liposomes is examined by using multilamellar liposomes prepared with egg phosphatidylcholine or dipalmitoylphosphatidylcholine and by varying the surface charge by addition of dicetyl phosphate. Investigations are performed by gel chromatography and electron microscopy. Glutarylphosphatidylcholine is in micellar form (rod-like micelles or globular micelles). The structures obtained depend on the fatty acid saturation of liposomes and on the charge of liposome (addition or not of dicetyl phosphate). With egg phosphatidylcholine/glutarylphosphatidylcholine dispersions, an aspect more similar to myelinic figures than liposomes is observed, while in the presence of dicetyl phosphate, liposomes similar to control egg phosphatidylcholine liposomes are obtained. Gel chromatography on Sepharose 4B and turbidity measurements prove that dicetyl phosphate increases the stability of egg phosphatidylcholine/glutarylphosphatidylcholine mixtures. On the other hand, in dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, incorporation of dicetyl phosphate destabilizes bilayer structure and the formation of mixed micelles occurs. Viscosity measurement shows, in the presence of dicetyl phosphate, an increased fluidity for dipalmitoylphosphatidylcholine/glutarylphosphatidylcholine dispersions, in agreement with the micellar organization. These data confirm that the disorganization of liposomal membranes by dicarboxylic phosphatidylcholine depends on the fatty acid composition of phosphatidylcholine and on the presence of dicetyl phosphate.     

Fetal-maternal secretion of prostaglandins in the cow

A study was conducted to measure the blood plasma concentrations of prostaglandin F₂α (PGF₂α), 13,14-dihydro-15-keto-prostaglandin F (PGFM), 6-keto-prostaglandin F₁α (6-keto), prostaglandin E₂ (PGE₂), and thromboxane B₂ (TBX₂) in the ovarian vein, uterine artery, uterine vein, umbilical artery and umbilical vein in 24 cows from days 80 to 260 of pregnancy. Blood was collected during surgery and all prostaglandins were measured using specific radioimmunoassay procedures. Results indicate that PGF₂α blood levels are higher in the umbilical vessels and uterine vein than in the ovarian vein and uterine artery. PGFM and PGE₂ showed a trend towards higher values in the umbilical than in the maternal vessels, but the levels of 6-keto and TBX₂ were not different among the vessels studied. No differences across time couls be observed in any of the prostaglandins measured, partly due to the great variability in blood levels among animals during the same stage of pregnancy.     

cAMP stimulates cholesteryl ester clearance to high density lipoproteins in J7774 macrophages.

The regulation by cAMP of cholesteryl ester hydrolysis and net depletion of cellular cholesteryl ester (cholesteryl ester clearance) in J774 murine macrophages was explored. Using Sandoz 58035 to selectively inhibit acyl CoA:cholesterol acyltransferase, we showed that the absolute rate of cholesteryl ester hydrolysis was stimulated 2-fold in J774 cells by the cAMP analogues 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate and dibutyryl-cAMP. The rate of hydrolysis was also stimulated by prostaglandin E1, by cholera toxin, and by a mixture of forskolin and isobutylmethylxanthine, but was not affected by epinephrine or dibutyryl-cGMP. These data demonstrate that cholesteryl ester hydrolysis in J774 cells can be stimulated by cAMP-dependent protein kinase. Cholesteryl ester clearance from J774 cells was achieved upon incubation with high density lipoproteins (HDL) plus CPT-cAMP but not with HDL alone. HDL-mediated cholesteryl ester clearance was dependent on the concentration of both HDL and CPT-cAMP. The data suggest that the defect responsible for the lack of HDL-mediated cholesteryl ester clearance in J774 cells involves a failure to modulate cAMP levels.     

Image cytometry of progesterone receptor expression during the cell cycle in the MCF-7 cell line

Progesterone receptors (PR) appear to be distributed in a heterogeneous way in mammary tumor cells. The study presented here was designed to examine if heterogeneity of PR expression is cell-cycle dependent. Immunofluorescence techniques were used to label PR on the MCF-7 human breast cancer cell line and image cytometry was used to analyze the PR expression during G0 (Ki-67 antigen-negative cells), G1, S, and G2/M cell-cycle phases. A second PR, BrdU, and DNA analysis was performed to study PR expression in the S-phase (BrdU-positive cells). Our results show that PR synthesis occurs preferentially during the G0-G1 transition and that PR levels are constant during the G1-G2 transition. The PR expression appears to be cell-cycle related and may therefore explain the heterogeneity of PR expression. However, the possibility that PR heterogeneity may be linked to the existence of PR-negative subclones cannot be ruled out.     

Cerebral Metabolic Effects of Monoamine Oxidase Inhibition in Normal and 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Acutely Treated Monkeys

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopaminergic cell death in the substantia nigra pars compacta (SNpc) and clinical parkinsonism in humans and experimental animals. Pretreatment with monoamine oxidase inhibitors prevents this cell death and associated parkinsonism by blocking the oxidation of MPTP to a toxic intermediate. The 2-deoxyglucose method was used to study the acute effects of MPTP in the monkey brain and the effects of monoamine oxidase inhibition on local cerebral glucose utilization in both normal and MPTP-treated monkeys. MPTP administration alone caused a major increase in glucose utilization in the SNpc and smaller increases in some subnuclei within the ventral tegmental area in which eventual dopaminergic cell loss also occurs. Pretreatment with pargyline abolished these metabolic increases, a finding suggesting both that the oxidized product of MPTP generates the metabolic increases and that the increased glucose consumption may contribute to cell toxicity. On the other hand, in most cortical, thalamic, striatal, brainstem, and cerebellar areas MPTP alone caused reductions in glucose utilization, and pargyline failed to prevent these effects. Pargyline alone depressed metabolism in the locus coeruleus and a few other monoaminergic structures.     

Water quality research in Loosdrecht lakes: the salient features

Ecosystem research by the working group Water Quality Research Loosdrecht lakes (WQL) was carried out from 1979 to 1990. A coordinated research programme, involving several research institutes and laboratories in The Netherlands, was initiated in 1983, i.e. a year before the reduction of external phosphorus loading by stripping, became effective. The paper summarizes the main results, with emphasis on insight they provide into the lake ecosystems.