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.     

1H- and13C-NMR spectroscopic studies of lipid extracts of the red algaGracilaria longa

Lipid extracts of the red algaGracilaria longa were studied by¹H- and¹³C-NMR spectroscopy. Peaks in the¹³C-NMR spectra attributable to sterols, chlorophylls and carotenoids allowed free and acylated cholesterol, chlorophylla and lutein to be identified as the most abundant components of these classes. A content of 0.5 ± 0.1 μmoles of total cholesterol/g wet alga was estimated from the¹H-NMR spectrum, which also allowed the determination of the phosphatidylcholine/total lipid molar ratio (9.5 ± 0.5%). The¹³C-NMR spectroscopic experiments provided information on the position of the double bonds on the fatty acid residues. A comparison between NMR spectra of lipid extracts obtained for wet and dried alga showed that the alga undergoes both a dramatic peroxidation and some glycolipid degradation during the drying process.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

Analysis of Chlamydomonas reinhardtii histones and chromatin

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.     

Human immunodeficiency virus protein gp120 interferes with β-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes

Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of³²P into a soluble acidic protein of 80,000M _r , which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to³H-myristic acid indicated that the acidic 80,000M _r protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M _r MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.     

High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry