Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Adipocyte cellularity in different adipose depots in bulls of seven Spanish breeds slaughtered at two body weights

The influence of body weight (BW) at slaughter and genotype on adipocyte size and number in the omental (OM), perirenal (PR), subcutaneous (SC) and intermuscular (IM) adipose tissues was studied in 168 bulls of Spain's local Asturiana, Avileña, Morucha, Parda Alpina, Pirenaica, Retinta, and Rubia Gallega cattle breeds. The young bulls were slaughtered at two BWs, 320 and 540 kg. The results obtained showed the higher amounts of lipids that accumulated between 320 and 540 kg BW (P < 0.001) to be ascribable primarily to adipose cell hypertrophy, i.e. larger adipocyte size, in the OM and PR depots (P < 0.001). In addition to hypertrophy, there was also an increase (P < 0.001) in the number of adipose cells, i.e. hyperplasia, in the SC and IM adipose depots. Significant differences were observed when comparing the different genotypes, with the Morucha, Retinta and Avileña breeds having the highest amount of adipose tissue and the largest adipocytes. The Asturiana and Rubia Gallega breeds had the lowest amount of adipose tissue and the smallest adipocytes. The Pirenaica and Parda Alpina breeds had intermediate values in between the two groups identified above. In short, the results were indicative of different lipid deposition patterns in the different breeds depending on the individual growth and maturation rates in each. Similar findings were made when comparing the different adipose tissue depots, with adipocyte hypertrophy being the main factor responsible for lipid accumulation in the OM and PR depots, as opposed to adipocyte hyperplasia in the SC and IM depots.     

Lipogenic activity in goats (Blanca celtibérica) with different body condition scores

The aim was to study the variation in the amount of adipose tissue, size and number of adipocytes and lipogenic enzyme activities in does with different body condition scores (BCS). These parameters were studied in the omental (OM), mesenteric (MES), perirenal (PR), subcutaneous (SC) and intermuscular (IM) fat depots of 22 adult, non-pregnant and non-lactating Blanca celtibérica does, with a BCS ranging from 1.5 to 4.5 (scale 0–5). SC adipose tissue showed the highest deposition-mobilization phase (highest fat relative to the variation per BCS unit of 74%), followed by OM (65%), PR (63%), MES (48%) and IM (46%) adipose depots. Adipocyte size varied with the amount of fat (P < 0.001), while the SC depot was the only depot that showed a positive correlation between adipocyte number and BCS (r² = 0.20; P < 0.05). The activities of glycerol 3-phosphate dehydrogenase (G3PDH) and fatty acid synthetase (FAS) enzymes were not generally affected by BCS, due to the fact that animals were on a maintenance diet prior to slaughter. The findings obtained in this work indicate that the processes of storage and mobilization of fat reserves in adult goats are principally due to variation in the size of the adipocyte cells. Therefore, the use of BCS could be a good method for predicting nutritional status in goats.     

Abrogation of the antiproliferative activity of oncostatin M by a monoclonal antibody.

Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.     

Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli

Biogenesis of the outer membrane (OM) in Gram‐negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well‐studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans‐envelope Tol‐Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol‐Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol‐Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol‐Pal complex may promote OM invagination during cell division.     

Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.     

Localized application of soil organic matter shifts distribution of cluster roots of white lupin in the soil profile due to localized release of phosphorus

Background and Aims

Phosphorus (P) is a major factor controlling cluster-root formation. Cluster-root proliferation tends to concentrate in organic matter (OM)-rich surface-soil layers, but the nature of this response of cluster-root formation to OM is not clear. Cluster-root proliferation in response to localized application of OM was characterized in Lupinus albus (white lupin) grown in stratified soil columns to test if the stimulating effect of OM on cluster-root formation was due to (a) P release from breakdown of OM; (b) a decrease in soil density; or (c) effects of micro-organisms other than releasing P from OM.

Methods

Lupin plants were grown in three-layer stratified soil columns where P was applied at 0 or 330 mg P kg⁻¹ to create a P-deficient or P-sufficient background, and OM, phytate mixed with OM, or perlite was applied to the top or middle layers with or without sterilization.

Key Results

Non-sterile OM stimulated cluster-root proliferation and root length, and this effect became greater when phytate was supplied in the presence of OM. Both sterile OM and perlite significantly decreased cluster-root formation in the localized layers. The OM position did not change the proportion of total cluster roots to total roots in dry biomass among no-P treatments, but more cluster roots were concentrated in the OM layers with a decreased proportion in other places.

Conclusions

Localized application of non-sterile OM or phytate plus OM stimulated cluster-root proliferation of L. albus in the localized layers. This effect is predominantly accounted for by P release from breakdown of OM or phytate, but not due to a change in soil density associated with OM. No evidence was found for effects of micro-organisms in OM other than those responsible for P release.     

Interleukin-6 signal transducer gp130 mediates oncostatin M signaling.

Oncostatin M (OM) is a multifunctional cytokine that is structurally and functionally related to interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). The specific receptor for OM has been demonstrated (by chemical cross-linking) to be a 150-kDa protein in a number of cell lines. The IL-6 signal transducer, gp130, is also an affinity converter for the LIF receptor. It does not bind to either IL-6 or LIF, but associates with the alpha subunits of the receptors and transduces the signals. We examined the possible involvement of gp130 in OM binding and signaling. We demonstrate that: (a) anti-gp130 monoclonal antibodies (mAbs) block the inhibitory effect of OM on A375 cell growth, (b) the binding and cross-linking of 125I-OM to H2981 cells are completely abolished by anti-gp130 mAbs, (c) the cross-linked OM-receptor complex is immunoprecipitated by anti-gp130 mAbs, and (d) COS-7 cells transfected with the full-length cDNA encoding gp130 exhibit increased OM binding and cross-linking, which are also blocked by anti-gp130 mAbs. Therefore, we conclude that the 150-kDa OM binding protein previously characterized in a variety of cell lines is gp130. OM is the natural ligand for gp130 and gp130 mediates the biological responses of OM.     

Disruptive effects of tris and sodium lauroyl sarcosinate on the outer membrane of Pseudomonas cepacia shown by fluorescent probes

The disruptive effects of Tris buffer and sodium lauroyl sarcosinate (Sarkosyl) on the outer membrane (OM) of Pseudomonas cepacia were investigated with several fluorescent probes. Tris increased the permeability of the OM to 6-anilino-l-naphthalenesulphonic acid and 2-p-toluidinylnaphthalene-6-sulphonate. The degree of damage to the OM was enhanced when the pH was decreased 3-(N-morpholino)propanesulphonic acid buffer had a small but significant effect at acid pH, while citrate/phosphate buffer showed insignificant effects. Sarkosyl released 3,3'-dipentyloxacarbocyanine iodide (CC5) from CC5-labelled OM or whole cells and altered OM fluidity as studied by fluorescence polarization.     

Organic matter distribution and retention along transects from hilltop to kettle hole within an agricultural landscape

In agricultural landscapes, the spatio-temporal distribution of organic matter (OM) varies greatly across landscape structures and soil types. We investigated patterns of organic carbon (OC) content, polyvalent cations, and isotopic values for specific OM fractions along transects spanning topographic positions from erosional to depositional areas, including aquatic sediments within a single kettle hole. We hypothesized different drivers exist at different scales. At the transect scale, we hypothesized (1) landscape form and land management to explain patterns of isotopic and OC content from different OM fractions. At the aggregate scale, (2) we expected different OM-mineral associations to explain stabilized OM. We also hypothesized, (3) that shallow sediment δ¹³C and δ¹⁵N of the kettle hole reflected different terrestrial sources. We found that distinct differences in the OM turnover rates existed between the fractions suggesting that different processes are affecting the transformation rates that are recorded in the isotopic composition patterns. Erosion along with plant productivity drive mineral-associated fractions over the transect, while microbial decomposition and slurry influence freely available and aggregated OM fractions. The type and magnitude of OM-mineral associations changed along the transect while binding OM of different decomposition status. OM in mineral-associated fractions in kettle hole sediments were derived from clay- and silt-sized particles from the field, whereas OM in freely available and aggregated fractions potentially originated from macrophytes. We conclude that kettle holes constitute important sinks for terrestrial OM across the landscape.     

Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells.

A stable Chinese hamster ovary (CHO) cell line producing high levels of human oncostatin M (OM) was generated by transfecting a heterologous gene coding for the protein. This novel construct was comprised of the gene for the transforming growth factor-beta 1 (TGF-beta 1) signal peptide fused to the gene for mature human OM. Amplification with methotrexate produced milligram quantities of this recombinant OM, which was processed correctly, glycosylated, and found to have biological functions similar to those of natural OM.     

Major outer membrane proteins: common antigens in enterobacteriaceae species

The major outer membrane (OM) proteins of 23 enterobacterial strains (principally clinical isolates) and five non-Enterobacteriaceae species were investigated by the sodium dodecyl sulphate-polyacrylamide gel immunoperoxidase (SGIP) technique to evaluate antigenic cross-reactivity among these proteins. All enterobacterial strains contained one or more peptidoglycan-associated major OM proteins, cross-reactive with the peptidoglycan-bound protein I of Escherichia coli, and one non-peptidoglycan-bound heat-modifiable protein, cross-reactive with protein II of E. coli. Results indicated that antigenic cross-reactivity of the major OM proteins is a general phenomenon in the family Enterobacteriaceae, independent of any molecular weight variation of the corresponding proteins in different bacterial strains. SGIP experiments carried out with OM preparations of other species showed no cross-reactivity of any of their OM proteins with enterobacterial major OM proteins. The significance of the immunological relatedness of OM proteins for the classification of some Enterobacteriaceae is discussed.     

Hexagonal periodicity in the outer membrane of Bacteroides buccae

In Bacteroides buccae, a hexagonally arranged periodic structure was found in the outer membrane (OM), in addition to hexagonal lattices present in its external surface layer (S-layer). This crystalline OM protein (COMP) was present as patches on the concave fracture face (the outer leaflet) of the OM in freeze-fractured cells. Occasionally, hexagonally arranged structures could also be seen on the convex fracture face of the OM as 'fingerprints' of the COMP. The OM proteins were isolated and analysed by gel electrophoresis. The major band protein had an apparent molecular mass of 17 kDa. Whether the minor band proteins are also components in the structure of the COMP remains to be elucidated. Other oral Gram-negative anaerobic rods studied did not show any periodicity in their OM.     

Mutations in enterobacterial common antigen biosynthesis restore outer membrane barrier function in Escherichia coli tol-pal mutants

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects the cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.     

The binding of Pseudomonas aeruginosa outer membrane ghosts to human buccal epithelial cells

The binding of outer membrane (OM) ghosts derived from Pseudomonas aeruginosa strain 492c to human buccal epithelial cells (BECs) was examined. Electron microscopic examination of the binding of OM ghosts to BECs revealed direct OM ghost-BEC interaction. Equilibrium analysis of the binding of OM ghosts to trypsinized BECs employing the Langmuir adsorption isotherm indicated the number of binding sites (N) to be 1.3 X 10(-4) micrograms protein per BEC with an apparent association constant (Ka) of 3.4 X 10(-2) mL/microgram protein. The Langmuir analysis of binding of OM ghosts to untrypsinized BECs was complex, suggesting two possible classes of receptors, a high affinity-low copy number class (Ka, 7.8 X 10(-2)mL/microgram protein; N, 8.6 X 10(-5) microgram protein per BEC) and a low affinity-high copy number class (Ka, 3.7 X 10(-3)mL/microgram protein; N, 9.2 X 10(-4)microgram protein per BEC). Sugar inhibition studies incorporating D-galactose enhanced binding to each BEC type. N-Acetylneuraminic acid and N-acetylglucosamine both enhanced binding of OM ghosts to untrypsinized BECs, while inhibiting binding to trypsinized BECs. D-Arabinose inhibited binding to both BEC types. Binding of OM ghosts to both BEC types was greatly inhibited by D-fucose, while L-fucose only greatly inhibited binding to untrypsinized BECs. These sugar inhibition data demonstrated a difference in the binding of OM ghosts to trypsinized and untrypsinized BECs and possibly reveal the nature of the receptor(s), free of possible bacterial metabolic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption of oligo-L-[35S]methionine after feeding of a low casein or a low soybean protein isolate diet in rats.

We studied the absorptive properties of oligo-L-methionine (OM), which is an enzymatically synthesized and slowly digestible peptide. Previously, we demonstrated that when OM was added to a low casein diet, the improvement of the body weight gain was higher than when OM was added to a low soybean protein isolate (SPI) diet and we suggested that the difference in the supplementary effect of OM depends on its absorptive rate. In the present study, the OM absorption estimated by the portovenous difference in radioactivity derived from 35S-labeled OM was higher in the casein diet than in the SPI diet in early stages of feeding after fasting. Absorbed OM was quantified by subtracting the radioactivity of [35S]OM remaining in the whole gut from the ingested [35S]OM, 90 and 180 min after feeding casein and SPI diets containing 3% [35S]OM. We also estimated the absorptive efficiencies by subtracting the amount of radioactivity remaining in the intestines from the amount of [35S]OM emptied from the stomach as percentages of the emptied OM. Both the amount of absorbed OM and absorptive efficiencies of OM were higher in the casein group than in the SPI group, and the higher absorptive efficiency in the casein group indicates a higher digestibility for OM when rats are fed a 3% OM diet after fasting. The digestibility of [35S]OM measured by fecal excretion of radioactivity of OM during normal feedings for diets containing 0.3% [35S] OM for 7 days was about 80% in the casein group and 60% in the SPI group. We conclude that the different supplementary effects of OM in the low casein and SPI diets depend on the difference in OM digestibility. The difference in the digestibility of OM may partly depend on the faster absorption rate of OM in the early stages of feeding.     

How complement kills E. coli. I. Location of the lethal lesion

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.     

The Fluidity of the Bacterial Outer Membrane Is Species Specific

The outer membrane (OM) is an essential barrier that guards Gram-negative bacteria from diverse environmental insults. Besides functioning as a chemical gatekeeper, the OM also contributes towards the strength and stiffness of cells and allows them to sustain mechanical stress. Largely influenced by studies of Escherichia coli, the OM is viewed as a rigid barrier where OM proteins and lipopolysaccharides display restricted mobility. Here the discussion is extended to other bacterial species, with a focus on Myxococcus xanthus. In contrast to the rigid OM paradigm, myxobacteria possess a relatively fluid OM. It is concluded that the fluidity of the OM varies across environmental species, which is likely linked to their evolution and adaptation to specific ecological niches. Importantly, a fluid OM can endow bacteria with distinct functions for cell-cell and cell-environment interactions.