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.     

Isolation of tomato cell lines with altered response to Fusarium cell wall components

Summary To obtain Tomato cell lines with an altered capacity to respond to heat-released cell wall components (elicitor) of a tomato pathogen (Fusarium oxysporum f. sp. lycopersici), positive and negative selection experiments, using BUdR enrichment techniques, were carried out on suspension cultures of the susceptible, low phytoalexin producer cultivar Red River. Both high and low phytoalexin producing clones were isolated. Further tests demonstrated that not all phytoalexin-producing clones were more susceptible to the elicitor toxic effect, and that they were altered also in the speed of response to fungal cell wall components. Cells selected with Fusarium elicitor showed the same behaviour when challenged by Phytophthora infestans elicitor, thus suggesting in this case lack of specificity. The results are finally discussed with a view to using the technique both as a tool to study the genetics and physiology of hostparasite interactions and as a possible new method for the selection of pathogen resistant genotypes.Paper no. 1224 IPRA-CNR; research supported by an EEC-BAP contract     

Fine structure of the receptors at the myotendinous junction of human extraocular muscles

The myotendinous junction of the human extraocular muscles was studied by electron microscopy. Some peculiar receptorial structures have been found in the majority of the samples examined. These structures are very small and consist of 1) the terminal portion of one muscle fibre, 2) the tendon into which it inserts and 3), within the tendon, a rich nerve arborization, whose branches are always very close to the muscle component. Only one discontinuous layer, made up of flat cells, which lack a basal lamina and often show pinocytotic vesicles, encapsules every musculo-tendinous complex. The tendinous component consists of amorphous ground substance of different electron density, of collagen and elastic fibres and is divided in compartments by ramified cells, which make an inner capsular-like covering to the nerve fibres. Three types of afferent nerve endings can be identified. One type is usually more frequent than the others, possesses a large number of neurotubules and neurofilaments and few mitochondria and is always surrounded by a Schwann cell which forms finger-like processes penetrating into the axoplasm. The second type is only partially enveloped by the Schwann cell. The axoplasm is devoid of neurotubules and contains few neurofilaments, several mitochondria and groups of small clear vesicles placed in the areas uncovered by the glial sheath. The third one is completely surrounded by the Schwann cell, but is devoid of neurotubules and neurofilaments and full of mitochondria. These morphological features correspond well with the probable role of these receptorial structures, which is to ensure very exact and precise ocular movements.     

Isolation and characterization of three protein proteinase isoinhibitors from the granular fraction of horse neutrophilic granulocytes

Three cathodically migrating protein protease isoinhibitors were isolated from the granule-rich fraction of equine neutrophilic granulocytes by means of FPLC chromatography, in addition to two previously described anodically migrating inhibitors. The three isoinhibitors had an identical enzyme specificity which was equal to the two previously described isoinhibitors; they inhibited exclusively proteinase K and subtilisin. The inhibitors retained their activity between pH 1 and 12. They also were heat stable at 100 degrees C for 20 min. Neither the biological function of isoinhibitors nor the fundamental role of granular protease inhibitors of such narrow and peculiar enzyme specificity are known.     

Isolation and characterization of two new low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of equine neutrophilic granulocytes

A new species of protein proteinase inhibitors was detected in the granule-rich fraction of equine neutrophilic granulocytes. Five isoinhibitors were identified with a narrow enzyme specificity towards two microbial proteinases, e.g., proteinase K and subtilisin. Two isoinhibitors were purified and partially characterized. They had an Mr of 11,300 and 7400, respectively, and were resistant to perchloric acid and heat treatment at 100 degrees C for 20 min. The inhibitors retained their activity over a broad range of pH (1-9 and 1-12, respectively). The possible biological function of this species of protein proteinase inhibitors as defensins (= endogenous antibiotics) is tentatively discussed.     

Puromycin photoaffinity labels small- and large-subunit proteins at the A site of the Drosophila ribosome

[3H]Puromycin covalently incorporates into the protein and to a much lesser extent into the RNA components of Drosophila ribosomes in the presence of 254-nm light. The photoincorporation reaction takes place with a small number of large- (L2 and L17) and small- (S8 and S22) subunit proteins as determined by two-dimensional gel analysis. More quantitative one-dimensional gel results show that puromycin reacts with each of these proteins in a functional site specific manner. The small percentage of the total labeling that occurs with rRNA also appears to be site specific. The rRNA labeling arises from a puromycin-mediated cross-linking of ribosomal protein and rRNA. Ionic conditions shift the pattern of puromycin-labeled ribosomal proteins. These results suggest that puromycin can occupy two distinct sites on Drosophila 80S ribosomes. The pattern of ribosomal proteins labeled by puromycin is affected by the presence of other antibiotics such as emetine, anisomycin, and trichodermin.     

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.