Influence of acid concentration and temperature on fixed chromatin during feulgen hydrolysis

Summary Labelled nucleic acid were extracted from fixed, air-dried smears of Ehrlich's ascites tumour cells by fractionated hydrolysis and measured by liquid scintillation. It was found that the rates of RNA and DNA depolymerisation and of DNA depurination depended on temperature in the same way. The DNA extraction patterns retained their form when the temperature was varied. When the hydrolysis was performed in decreasing acid concentrations, however, there was a concomitant change in the form of the depolymerisation pattern. This change affects the amount of aldehyde groups available for dye-binding with the Feulgen method after the optimal hydrolysis time. The alteration in shape of the Feulgen curve is discussed and supposed to be due to an increased interaction between DNA and other macromolecules. It is suggested that this interaction may be useful in detecting differences in chromatin stability between cells which differ in gene activity.     

Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each individual subtype in heterologous systems. We report here the transfection, selection and preliminary pharmacological characterization of two mammalian cell lines, adherent Shionogi S115 mouse mammary tumour cells and human B-lymphoblastoid IBW4 cells growing in suspension, expressing the human alpha 2-AR subtypes alpha 2-C4 and alpha 2-C10 at densities of approx. 2 x 10(5) receptors/cell. Transfection of the subtype genes was verified using a specific RNase protection assay. Pharmacological characterization was carried out with [3H]rauwolscine binding, which was inhibited by oxymetazoline and prazosin in a subtype-selective manner. The sensitivity of (-)-noradrenaline binding to the GTP-analogue 5'-guanylylimidodiphosphate suggested that the receptors are coupled to G-proteins. This was verified in S115 cells by efficient inhibition of forskolin-stimulated cAMP production by the alpha 2-AR agonists, (-)-noradrenaline and clonidine. These cell lines thus appear to be suitable for pharmacological studies on receptor function and ligand binding.     

[3H]Citalopram Binding to Brain and Platelet Membranes of Human and Rat

Citalopram, a selective serotonin (5-HT) uptake inhibitor with antidepressant properties, was found to bind with high affinity to the 5-HT transporter from human neuronal and platelet membranes. At 20 degrees C, KD was about 1.5 nM in both tissues. [3H]Citalopram bound to rat neuronal membranes with higher affinity than to human neuronal and platelet membranes; at 20 degrees C KD was about 0.7 nM. The Bmax value for the binding of [3H]citalopram to platelet membranes was close to that found using the 5-HT uptake inhibitors [3H]imipramine and [3H]paroxetine, suggesting that all three 5-HT uptake inhibitors bind to the 5-HT transporter. The dissociation rate of [3H]citalopram increased twofold with each 4-5 degree C increase in temperature in both human and rat membranes, although at any given temperature, the dissociation rate was about four times faster in the human neuronal and platelet membranes than in rat neuronal membranes.     

Expression of the outer membrane protein P.69 ofBordetella pertussis inBacillus subtilis

Summary The gene coding forBordetella pertussis P.69 protein was cloned and expressed inBacillus subtilis. The expression vector contained the promoter region and the sequence coding for the whole or truncated signal sequence of the -amylase gene fromB. amyloliquefaciens. Using either construction the level of expression was relatively low and the protein was found in the particulate fraction. The protein migrated in gel electrophoresis slower than expected from its deduced amino acid content thereby giving the appearance of having an anomalously large molecular mass.     

Morphogenesis of the hatching gland of Atlantic halibut (Hippoglossus hippoglossus)

Summary The halibut hatching gland (HG) cells are first observed as a cellular disc in front of the embryonic head around the midpoint of intra ovo development. The disc is subsequently transformed into a loop of increasing diameter as the HG cells migrate over the anterior part of the yolk sac. When the HG disc is transformed into a loop, the density of HG cells is highest at the migratory front. Some HG cells lag behind the migrating front at the early stages of HG development. At maturity, all cells are contained in a narrow belt which is about 10 cells wide. The HG belt structure consists of a monolayer of HG cells, and is maintained while the cells migrate between the two epidermal cell layers. Migration is halted about 2 days before normal hatching when the HG cells reach a destination at about a right angle to on the embryonic axis. Under the scanning electron microscope, the differentiating HG cells protrude as a ridge the yolk sac surface. The HG cells immunostain with antiserum to hatching enzyme when the HG is observed as a crescent structure around the embryonic head. By counting the number of immunostaining cells in composite photos of the entire yolk sac membrane, we found that the HG belt consists of approximately 2000 secretory cells at maturity. This cell number stays fairly constant throughout the period of HG cell migration. Accordingly, mitoses of the halibut HG cells have generally ceased prior to morphogenesis, and cytodifferentiation is already quite advanced when cell migration starts. Offprint requests to: J.V. Helvik     

Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly inEscherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzmes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.