Expression of recombinant human granulocyte colony-stimulating factor in CHO dhfr- cells: new insights into the in vitro amplification expression system.

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.

Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

Plasma pancreatic polypeptide response to secretin.

OBJECTIVE: Intravenously administered secretin stimulates pancreatic polypeptide (PP) release in patients with endocrine enteropancreatic tumors, but data in patients with nontumorous disorders are controversial. Therefore, we aimed to evaluate the plasma PP pattern after secretin administration in healthy subjects and in patients with gastroduodenal diseases investigated for recurrent ulcer disease and/or hypergastrinemia. METHODS: Synthetic secretin was given as an intravenous bolus (2U/kg) in ten patients with Zollinger Ellison syndrome, ten with duodenal ulcer, ten with atropic gastritis and ten healthy volunteers. Blood samples were taken before and at regular intervals for 30min after secretin injection. Plasma PP and gastrin levels were measured by radioimmunoassay. RESULTS: Secretin promptly and significantly (P<0.01) increased PP plasma levels in all groups of subjects without any differences in peak values. There were no significant correlations between PP and gastrin plasma levels. CONCLUSIONS: Secretin at pharmacological doses is a powerful stimulus for PP release.

An in vitro amplification approach for the expression of recombinant proteins in mammalian cells.

A method for the expression of recombinant DNA products in mammalian cells based on in vitro amplification of gene units is described. Gene cassettes containing either a selectable marker or the gene of interest are mixed at different molar ratios, and linear polymers are formed using forced head-to-tail ligation. After introduction of the polymers into mammalian cells, transformants with various amounts of the amplified gene unit are obtained. For a first characterization of the system, the gene coding for chloramphenicol acetyltransferase (CAT) has been used to produce polymers containing a single neomycin-resistance gene ligated to different numbers of CAT gene units and used for transfection into HeLa cells. All isolated G418 (gentamycin)-resistant cell transformants which received in vitro amplified DNA were found to express high levels of CAT activity in a stable manner. Southern-blot analysis of individual clones revealed multiple copies of the gene integrated head-to-tail in the genome. This system allowed us to express the gene coding for human prepro-endothelin-1 in A617 human vascular-smooth-muscle cells. The recombinant protein was shown to be correctly processed and biologically active endothelin-1.

[DMFT index values in patients treated for leukemia during growth]. / Valori dell'indice DMFT in soggetti trattati per leucemia nell'eta evolutiva.

Patients affected by acute leukemia under radiotherapy and chemotherapy, less than 12 years old were investigated about DMFT index. Patients were in total remission. All erupted permanent teeth, with attention to an eventual oligodontia of non erupted teeth also, excluding third molars, were investigated by clinical and X-Ray examination. Authors concluded that there is no difference between these patients and normal subjects for this index.

The physical state of a meal affects hormone release and postprandial thermogenesis.

There is evidence that food consistency may influence postprandial physiological responses. Recently we found that homogenization of a vegetable-rich meal significantly delayed the gastric emptying rate and was more satiating than the same meal in solid-liquid form. In this present study we investigated whether homogenization also influences endocrine and metabolic responses to the meal. Eight healthy men, aged 21-28 (mean 24.5) years, were given the meal (cooked vegetables 250 g, cheese 35 g, croutons 50 g and olive oil 25 g, with water 300 ml; total energy 2.6 MJ) in both solid-liquid (SM) and homogenized (HM) form, in random order, at 1-week intervals. Variables assayed were plasma glucose, insulin and glucose-dependent insulinotropic peptide (GIP) levels for 2 h and diet-induced thermogenesis (DIT) for 5 h. Plasma glucose pattern was similar after both meals. However, HM induced significantly greater insulin, GIP and DIT responses than SM. Mean integrated areas under the curves (AUC) were 1.7 (SEM 0.38) v. 1.2 (SEM 0.33) U/l per 120 min (P = 0.005) for insulin, 19.9 (SEM 2.44) v. 16 (SEM 1.92) nmol/l per 120 min (P = 0.042) for GIP, and 237.7 (SEM 16.32) v. 126.4 (SEM 23.48) kJ/300 min (P = 0.0029) for DIT respectively. Differences between GIP-AUC after HM and SM correlated significantly with differences between insulin-AUC after HM and SM (r2 0.62, P = 0.021). These findings demonstrate that homogenization of a meal results in a coordinated series of changes of physiological gastroentero-pancreatic functions and confirm that the physical state of the meal plays an important role in modulating endocrine and metabolic responses to food.

Postprandial gut peptide plasma levels in women with idiopathic slow-transit constipation.

BACKGROUND: As abnormalities of circulating gut regulatory peptides may have pathogenetic relevance in chronic idiopathic slow-transit constipation, we measured fasting and postprandial levels of plasma pancreatic polypeptide, motilin, cholecystokinin, neurotensin, and somatostatin in women with the disease. Results were compared with those of women with normal bowel habits. METHODS: Eight women with slow-transit constipation and 10 healthy women were studied. Blood samples were taken at regular intervals in fasting conditions and for 3 h after a standard solid-liquid meal (550 kcal). Gut peptide plasma levels were measured with a radioimmunoassay. RESULTS: Fasting gut peptide levels and postprandial pancreatic polypeptide responses were normal in constipated patients, in whom, however, motilin levels did not increase after the meal, and postprandial concentration-time curves of cholecystokinin, neurotensin, and somatostatin were delayed. Mean +/- standard error of the mean peak times in patients and in controls were, respectively, 99 +/- 14.7 and 46 +/- 4.1 min (P < 0.01, Mann-Whitney test) for cholecystokinin, 135 +/- 9.8 and 60 +/- 3.9 min (P < 0.01) for neurotensin, and 111 +/- 17.7 and 51 +/- 6.0 min (P < 0.05) for somatostatin. CONCLUSIONS: Patients with slow-transit constipation have abnormal postprandial patterns of motilin, cholecystokinin, neurotensin, and somatostatin.

A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.

EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons.