Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.     

Engineering crops, a deserving venture.

Plant transformation has had a deep impact on several aspects of basic and applied research. Genetic transformation has offered new opportunities compared to traditional breeding practises since it allows the integration into a host genome of specific sequences leading to a strong reduction of the casualness of gene transfer. One of the first target areas was plant protection against pests, pathogens and environmental stresses while the recent plant engineering programs are aimed at increasing food quality, in particular at increasing nutritional characteristics of food crops. Moreover, transgenic plants, tissue or cell cultures represent an attractive biological system for producing heterologous proteins since they offer economic and qualitative benefits. High yield production can be obtained and large-scale commercial production will take advantage of the existing infrastructure for crop cultivation, processing and storage. There are also qualitative benefits since protein synthesis secretion and post-translational modifications are similar in plants and animal cells. There are no human viral pathogens harboured by plants: thus, especially for pharmaceuticals, plants represent the safer production system. Plant transformation has become an essential instrument also for basic research, in particular for the functional characterisation of genes identified by sequencing of whole genomes. Large collections of insertion mutants have been obtained in the model plant Arabidopsis to provide a high level of genome saturation that means 95% chance of inactivating any gene at least once. To instil greater public confidence in modern plant biotechnology recent advances have already been made to overcome the potential risks for human health and environment.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Compartmentation of newly synthesized phosphatidylethanolamine in rat brain microsomes

Summary The compartmentation of the phosphatidylethanolamine newly synthesized in brain microsomesin vitro either by base exchange or net synthesis has been studied, using difluorodinitrobenzene as a chemical probe. The experimental results demonstrate that in rat brain microsomes the phosphatidylethanolamine molecules synthesized by base exchange and the bulk membrane lipid belong to different pools. Ca²⁺ bound to microsomes seems to be involved in the maintenance of the compartmentation of phosphatidylethanolamine. In the presence of Ca²⁺ the newly synthesized phosphatidylethanolamine molecules react with difluorodinitrobenzene as though they are organized in clusters. After biosynthesisin vivo orin vitro through the cytidine pathway, the compartmentation of the newly formed phosphatidylethanolamine appears less marked than after the synthesis through base exchange.     

Effect of various drugs producing convulsive seizures on rat brain glycerolipid metabolism

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-³H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3–60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciabily vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This lipid effect was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.     

Fusion of liposomes and rat brain microsomes examined by two assays

Summary Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb³⁺ or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R₁₈) to test fusion with the R₁₈ assay. The addition of either Ca²⁺ or Mg²⁺ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg²⁺ is similar to that elicited by Ca²⁺ if assessed with R₁₈, but much higher if determined by Tb-DPA. The Ca²⁺-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internal to vesicles. The contrary is true for the Mg²⁺-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to load microsomes with DPA. This allows the use of the Tb-DPA assay for testing the fusion of rat brain microsomes. Reconstituted microsomes fuse with liposomes. This fusion has characteristics similar to those of liposome-liposome fusion. However, no microsome-microsome fusion could be detected with either method. The two methods give different results, owing to the chemical properties of the assays. Indeed Tb-DPA implies the retention of vesicle content, whereas this is not required by the R₁₈ assay.     

Molecular Cloning of the N-Terminus of GTBP

Defects in mismatch repair genes cause the genetic instability characteristic of hereditary nonpolyposis colorectal cancer and a subset of sporadic colon tumors. The newest member of the mismatch repair gene family,GTBP, has recently been identified as a partial cDNA. Here, we describe the isolation of its 5′ terminus, allowing definition of the entire coding region. Several polymorphisms within the 5′ end were identified and are presented.     

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5 end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.     

Nerve growth factor antibodies recognize neurotrophin-3

The immunological properties of the neurotrophins NGF, BDNF, and NT-3 were compared using polyclonal and monoclonal antibodies against the subunit of mouse NGF. Affinity-purified anti-NGF IgG consistently recognized NGF and NT-3 on Western blots, and inhibited the trophic activity of NGF and NT-3 but not BDNF. In contrast, anti-NGF monoclonal antibodies did not block the trophic activity of either NT-3 or BDNF. These results are consistent with the greater structural overlap between NGF and NT-3 than between NGF and BDNF.