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.     

Salmonella serotypes isolated from human enteric processes in Recife, Pernambuco, during 1978-1980

From 13,196 faecal cultures made in Recife-Pernambuco during the period from 1978 to 1980, 1,720 strains of Salmonella were isolated. Serological typing on 1,387 of the isolates recognized 63 serotypes, 73.18% of which belonged to group B. The prevalent serotypes adding up to 1,231 strains (88.75% of the total of the isolates) were: S. typhimurium, S. saint-paul, S. poona, S. derby, S. agona, S. newport, S. oranienburg, S. infantis, S. tshiongwe and S. ndolo.     

Cellular location of the cleavage event of the polymeric immunoglobulin receptor and fate of its anchoring domain in the rat hepatocyte.

Transcytosis of polymeric immunoglobulin (pIg) across glandular and mucosal epithelia is mediated by a member of the immunoglobulin supergene family, the pIg receptor. During transcellular routing, the receptor is cleaved and its ectoplasmic domain, known as secretory component (SC), is released into secretions bound to pIg. Using receptor-domain-specific antibodies, we have combined cell fractionation and immunoblotting of rat liver to examine the cellular routing of the receptor, the cellular location of the cleavage event and the fate of the anchor domain. Cleavage is a late event in receptor processing. It appears to occur at the canalicular plasma membrane, since intact receptor is present in this membrane domain and no SC is detected in whole liver homogenate or in cell fractions. The membrane anchor remaining after cleavage can be recovered in bile, as well as in a low-density fraction obtained after equilibrium centrifugation of liver (microsomal fractions) on sucrose density gradients. These data suggest that the membrane-anchor domain may be internalized as well as secreted together with SC into bile.     

Purification and characterization of recombinant human interleukin 4. Biological activities, receptor binding and the generation of monoclonal antibodies.

A synthetic gene coding for human interleukin 4 (IL-4) was cloned and expressed in Saccharomyces cerevisiae (baker's yeast) as a C-terminal fusion protein with the yeast prepro alpha-mating factor sequence, resulting in secretion of mature IL-4 into the culture medium (0.6-0.8 micrograms/ml). A protocol was developed for purification of this protein. Crude cell-free conditioned medium was passed over a concanavalin A-Sepharose affinity column; bound proteins were eluted and further purified by S-Sepharose Fast Flow cation exchange and C18 reverse-phase h.p.l.c. Highly purified IL-4 was obtained by this method (0.3-0.4 mg per litre of culture) with a recovery of 51%. Thermospray liquid chromatography-mass spectrometry showed the C-terminal N-glycosylation site to be largely unmodified, and also showed that the N-terminus of the purified recombinant IL-4 (rIL-4) was authentic. Thiol titration revealed no free cysteine residues, implying that there are three disulphide groups, the positions of which remain to be determined. We have characterized the biological activities of the purified rIL-4. This material is active in B-cell co-stimulator assays, T-cell proliferation assays and in the induction of cell-surface expression of CD23 (the low-affinity receptor for IgE) on tonsillar B-cells. Half-maximal biological activity of the rIL-4 was achieved at a concentration of 120 pM. We have radioiodinated rIL-4 without loss of biological activity and performed equilibrium binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding studies on Raji cells, a human B-cell line. The 125I-rIL-4 bound specifically to a single class of binding site with high affinity (Kd = 100 pM) and revealed 1100 receptors per cell. Receptor-ligand cross-linking studies demonstrated a single cell-surface receptor with an apparent molecular mass of 124 kDa. Two monoclonal antibodies have been raised to the human rIL-4, one of which blocks both the biological activity of rIL-4 and binding to its receptor.     

Equalization of Z and W axes in chicken and quail oocytes.

The different morphological types of ZW pairs have been classified in three main types according to the relative extension of the free segment of the Z axis: 1, "long asynaptic segment;" 2, "medium asynaptic segment;" and 3, "equalized." Pre- and post-pairing types have also been defined. Frequencies of each type were determined at day 20 and day 21 of incubation, and one and three days after hatching. The changing frequencies and the morphological transitions observed show a definite sequence of ZW types that can be used as a timetable for pachytene substaging. Measurements made on each ZW type show that the Z axis of the chicken shortens from 20.6 microns to 13.1 microns. This shortening occurs both in the free segment (at a higher rate) and in the paired segment (at a lower rate). The synaptonemal complex becomes elongated while adjustment occurs. The equalized Z axis makes many twists around the W axis. However, a segment 1 micron long from the synaptic terminus is free from twists and is assumed to be the homologously paired region. The ZW pair of the quail shows a similar behavior but equalization of the Z and W axes ends earlier and forms a straighter synaptonemal complex as compared with the chicken. In both species a recombination nodule is strictly localized near the synaptic terminus. In the ZW pair of the quail the average location of this nodule is 0.14 microns from the synaptic terminus. The meiotic behavior of ZW pairs in birds may be conserved.     

Synaptic behaviour and recombination nodules in the human XY pair

A sample of 90 XY pairs from men with normal karyotypes has been analyzed by measuring their morphological features in electron micrographs of microspread spermatocytes. The classification of human XY types (Solari, 1980) has been given stricter definitions. Stepwise splitting of the axes is seen in types 1 and 2. The development of axial branches and lenhthening of the X axis is seen in type 3. In the two subtypes a and b of type 4 the net-like filamentous array grows in length to a maximum (average=59.7 m) in subtype b. The location of the putative Y kinetochore defines a short arm that measures 22.34% of Y axis length, and the kinetochore of the X axis defines a short arm of 38.15% of the axial length. The average number of excrescences in the X axis is 19.9 and in the Y is 4.3. The frequency of a non-homologous, distal end-joining grows steadily from type 0 to type 3. The average length of the synaptonemal complex (SC) in 51 XY pairs of types 1 and 2 is 1.33 m (SD=0.65) and it corresponds to 25.54% of the Y axis length. Thus, the average SC covers the short arm of the Y and the pericentromeric region. Maximum lengths of this SC may reach up to 81.8% of the Y axis, 30 recombination nodules (RNs) were located in 26 XY pairs, and 90% of the nodules are located in the distal half of the short arm of the Y axis. Thus, RNs are restricted to a segment much shorter than the length of the average SC. A gradient of decreasing probability of recombination may reach up to the centromeric region of the Y chromosome. Some possible consequences of these facts are discussed.     

Distribution and processing of the polymeric immunoglobulin receptor in the rat hepatocyte: morphological and biochemical characterization of subcellular fractions

The transepithelial transport of polymeric immunoglobulins is an essential process in the mucosal immune system. Transport across the epithelial cells of mucous or exocrine glands is affected by an integral membrane glycoprotein receptor known as membrane secretory component (SCm) or as polymeric immunoglobulin receptor (pIgR). This receptor binds polymeric immunoglobulins at the basolateral cell surface and mediates their transcellular translocation and their release from the apical plasma membrane into external secretions. Release depends on cleavage of the membrane-anchoring domain of the receptor, resulting in liberation of polymeric immunoglobulin bound to the ectoplasmic domain of the receptor (secreted SC or SCs) into extracellular secretions. Using a monoclonal antibody directed against the cytoplasmic tail of the receptor and a polyclonal antibody directed against the secreted ectoplasmic domain, we have combined cell fractionation and Western blotting techniques to examine the fate of these receptor domains in the hepatocyte. In this study, we characterize biochemically and morphologically the various subcellular components separated by our fractionation scheme, and correlate this with biochemical analysis of the receptor in each fraction.     

In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

Identification and distribution of two forms of the interleukin 1 receptor

Using affinity crosslinking techniques, we have biochemically characterized the interleukin-1 (IL1) receptor and investigated its distribution on a range of murine and human cell lines. We show that two forms of IL1 receptor can be identified on the basis of specific crosslinking with 125I-IL1 alpha and 125I-IL1 beta. The two receptor forms have an approximate molecular mass of approximately 80 and approximately 60 kDa, and were found on both murine and human cells. Their relative distribution shows no clear cell lineage restriction and does not correlate with preferential binding of IL1 alpha or IL1 beta. Some cells, such as the T helper cell line D10.G4.1, express both forms of the receptor. Iodine 125-IL1 was crosslinked to the two receptor forms and a partial peptide map analysis of the two receptor/ligand complexes was performed. Comigration of the major partial peptide fragments suggests that the approximately 80 and approximately 60 kDa forms of the receptor may be differentially processed forms of the same protein. Treatment of the approximately 60 kDa IL1 receptor on Raji cells with N-glycanase reduced its molecular mass by 12 kDa, showing that this lower molecular mass form is a glycoprotein; glycosylation differences alone probably do not account for the difference in mass between the two forms.