Characterization in primary monolayer culture of separated cell types from rabbit endometrium

Epithelial cells and stromal cells of the rabbit endometrium were separated by successive enzymic digestion of the uterine mucosa. Isolated cell types were obtained in high yield, with good viability, and were maintained in monolayer cultures for up to 2 weeks. Epithelial cells in monolayers appeared as polygonal cells, displayed contact inhibition, and showed the presence of microvilli on the cell surface, with many desmosomes. Stromal cells grew rapidly to confluence, displayed overgrowth, and had a fibroblastic appearance with an absence of junctional complexes between cells. Indirect immunofluorescence showed uteroglobin on the surface of epithelial but not of stromal cells, and only epithelial cells secreted uteroglobin into the medium. These results confirm the identity of the cells and provide biochemical evidence for the epithelial cellular origin of uteroglobin. The method allows the culture of separate endometrial cell types, which retain their morphology and differentiated function in vitro.     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.     

The reactivity of plasma phospholipids with lecithin:cholesterol acyltransferase is decreased in fish oil-fed monkeys

The size of low density lipoproteins (LDL) is strongly correlated with LDL cholesteryl ester (CE) content and coronary artery atherosclerosis in monkeys fed cholesterol and saturated fat. African green monkeys fed 11% (weight) fish oil diets have smaller LDL and less CE per LDL particle than lard-fed animals. We hypothesized that this might be due to a lower plasma lecithin:cholesterol acyltransferase (LCAT) activity in fish oil-fed animals. Using recombinant particles made of egg yolk lecithin-[14C]cholesterol-apoA-I as exogenous substrate, we found no difference in plasma LCAT activity (27 versus 28 nmol CE formed per h/ml) of fish oil- versus lard-fed animals, respectively; furthermore, no diet-induced difference in immunodetectable LCAT was found. However, plasma phospholipids from fish oil-fed animals were over 4-fold enriched in n-3 fatty acids in the sn-2 position compared to those of lard-fed animals. Additionally, the proportion of n-3 fatty acid-containing CE products formed by LCAT, relative to the available n-3 fatty acid in the sn-2 position of phospholipids, was less than one-tenth of that for linoleic acid. The overall rate of LCAT-catalyzed CE formation with phospholipid substrates from fish oil-fed animals was lower (5-50%) than with phospholipid substrates from lard-fed animals. These data show that n-3 fatty acids in phospholipids are not readily utilized by LCAT for formation of CE; rather, LCAT preferentially utilizes linoleic acid for CE formation. The amount of linoleic acid in the sn-2 position of plasma phospholipids is reduced and replaced with n-3 fatty acids in fish oil-fed animals. As a result, LCAT-catalyzed plasma CE formation in vivo is likely reduced in fish oil-fed animals contributing to the decreased cholesteryl ester content and smaller size of LDL particles in the animals of this diet group.     

Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.     

Characterisation of Antibodies Specific for Chick Brain Neural Cell Adhesion Molecules Which Cause Amnesia for a Passive Avoidance Task

Abstract: Antisera were prepared against six postsynaptic density glycoprotein fractions (150–180, 62–80, 50, 41, 33, and 28 kDa) that show enhanced fucosylation during memory formation after training day-old chicks in a one-trial passive avoidance task. Each antiserum was tested for its possible effect on memory retention. Bilateral intracranial injections of two of the antisera, R-1 and R-6, or their IgGs (IgG-1 and IgG-6), resulted in amnesia for the passive avoidance task when chicks were tested 24 h later. IgG-1 and IgG-6 antibodies were amnestic only when injected 5.5 h after training, and had no effect when injections were made 30 min before training, thus resembling an effect previously observed with polyclonal or monoclonal anti-N-CAM antibodies. IgG-1 and IgG-6 antibodies were found to be specific for protein epitopes of glycoproteins that contain a high amount of N-linked mannose and fucose, and a very low amount of polysialic acid and O-linked galactose. Absorption of IgG-6 antibodies with neural cell adhesion molecule (N-CAM) isolated from synaptic plasma membranes derived from day-old chick brain resulted in loss of amnestic effect. As we have previously shown that long-term memory for the passive avoidance task requires two waves of glycoprotein synthesis, the first occurring immediately after training and the second 5–8 h later, the present results suggest strongly that isoforms of N-CAM molecules with a low level of sialic acid are involved specifically in the establishment of an enduring memory for the experience of the passive avoidance task in chicks, possibly by stabilising changes in synaptic connectivity that encode the memory.     

Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%). This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.     

Goats and garbage in Khartoum,Sudan: A study of the urban ecology of animal keeping

Waste management in developing world urban areas is an acute problem which is linked to high growth rates and abatement methods less advanced than in the developed world. Existing neighborhood waste management practices often include the use of organic waste as a feed supplement for urban livestock (mainly goats, but also sheep and cattle). These urban animals bring economic benefits to less affluent residents, who would suffer an economic loss if their supply of urban waste was reduced or eliminated by an organized waste collection and disposal strategy. This paper employs a survey of 319 Greater Khartoum households to derive an estimate of waste intake per urban animal, and illustrate demographic factors (e.g., rural or urban roots, income, education, number of children) which influence animal keeping. A loglinear/logit model is developed to predict animal-keeping households. This model can be applied by waste management planners to estimate the frequency of urban animals in developing world urban areas.     

Endogenous ligands of rat lung beta-galactoside-binding protein (galaptin) isolated by affinity chromatography on carboxyamidomethylated-galaptin-Sepharose.

Rat lung beta-galactoside-binding protein (galaptin) is developmentally regulated during postnatal lung development. In common with other vertebrate galaptins, it is very labile when purified and dependent on the presence of exogenous thiol reagents. Reaction of rat lung galaptin with iodoacetamide resulted in a stable active carboxyamidomethylated galaptin that could be coupled to Sepharose. The resultant affinity matrix bound asialoglycoproteins, and these could be quantitatively eluted with disaccharide haptens. The carboxyamidomethylated-galaptin-Sepharose affinity matrix was used to search for endogenous ligands in 13-day-rat lung. Cytosolic fractions of developing rat lung contained no moieties that could be specifically eluted with disaccharide hapten. Only when membranous fractions were extracted with 1% Triton were glycoproteins solubilized that bound to the affinity matrix and could be specifically eluted with disaccharide hapten. The eluted glycoproteins were potent inhibitors of galaptin binding to asialo-orosomucoid. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis identified these glycoproteins as being of high Mr, with three components of Mr 160000-200000 and a smaller component of Mr 75000. This is the first evidence for specific membrane-associated glycoproteins being the ligands of rat lung galaptin.