A conditionally immortalized cell line model for the study of human prostatic epithelial cell differentiation

In the normal human prostate, undifferentiated proliferative cells reside in the basal layer and give rise to luminal secretory cells. There are, however, few epithelial cell lines that have a basal cell phenotype and are able to differentiate. We set out to develop a cell line with these characteristics that would be suitable for the study of the early stages of prostate epithelial cell differentiation. We produced a matched pair of conditionally immortalized prostate epithelial and stromal cell lines derived from the same patient. The growth of these cells is temperature dependent and differentiation can be induced following a rise in culture temperature. Three-dimensional co-cultures of these cell lines elicited gland-like structures reminiscent of prostatic acini. cDNA microarray analysis of the epithelial line demonstrated changes in gene expression consistent with epithelial differentiation. These genes may prove useful as markers for different prostate cell types. The cell lines provide a model system with which to study the process of prostatic epithelial differentiation and stromal-epithelial interactions. This may prove to be useful in the development of differentiation-targeted prostate cancer therapies.     

Cellular features of the extra-embryonic endoderm during elongation in the ovine conceptus

The extraembryonic endoderm of the elongating ovine conceptus was analyzed by scanning and transmission electron microscopy and by whole mount actin staining and immunofluorescence. Morphological and functional differences between the visceral endoderm (VE), the founding cell layer, and the parietal endoderm (PE) are presented. During the elongation process, the PE differentiated to fusiform multinucleated cells aligned parallel to the elongation axis of the conceptus, whereas the VE cells retained the aspect of typical epithelial cells. The multinucleated PE cells however, expressed cellular and nuclear markers typical of endodermal and polarized epithelial cells. The proteins of the extracellular matrix, laminin, and fibronectin, were specifically expressed in the PE. The presence of pairs of nuclei linked by mid-bodies positively stained with tubulin antibodies, indicated that the syncytial differentiation of the PE was due to karyokinesis which was not followed by cytokinesis rather than by cell fusion.     

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.     

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

Redressing the sex imbalance in knowledge of vector biology

The recent development of transgenic mosquitoes that are resistant to infection by the Plasmodium malarial parasite is a promising new tool in the fight against malaria. However, results of large-scale field releases of alternatively modified mosquitoes carried out during the 1970s and 1980s suggest that this approach could be difficult to implement in the field. These past attempts to control mosquito populations largely floundered as a result of our insufficient understanding of the behavioural ecology of released males. In spite of this, contemporary research on genetic control strategies has concentrated predominantly on molecular aspects, with little progress being made toward resolving key ecological uncertainties, male mosquito ecology being the most important. Here, we review the state of knowledge of male mosquito ecology, and highlight priorities for further research. Case studies of two crop pests, the Mediterranean fruit fly and melon fly, are given as examples of how knowledge of male ecology facilitates successful control in other species. Unless similar information becomes available for mosquitoes, any future genetic control strategy will risk failure.     

Leptin inhibits swallowing in rats

Swallowing is under the control of premotoneurons located in the medullary solitary tract nucleus. Although rats with transected midbrain do not seek out food, they are able to ingest food present near the mouth, and acute food deprivation induces an increase in food intake. Leptin is a satiety signal that regulates feeding behavior. Because leptin receptors are found within the caudal brainstem, and because food intake is regulated in midbrain transected rats, this study tested the hypothesis that leptin is able to modify the activity of premotoneurons involved in swallowing. Leptin was microinjected at the subpostremal level of the medullary solitary tract nucleus in anesthetized Wistar rats. Electromyographic electrodes in sublingual muscles allowed recording of swallowing induced by stimulation of sensitive fibers of the superior laryngeal nerve. Repeated stimulation induced rhythmic swallowing. Microinjection of leptin (0.1 pg and 0.1 ng) in the swallowing center induced an inhibition of rhythmic swallowing (latency of <30 s) as shown by the reduced number and strength of electromyographic activities, which could last several minutes. The threshold of the leptin-induced inhibition was close to 0.1 pg. Interestingly, the inhibitory effect of leptin was not observed in leptin receptor-deficient Zucker rats. Here we show that, in Wistar rats, leptin already known to modulate the discharge of medullary solitary tract nucleus-sensitive neurons involved in satiety reflexes can also modify the activity of swallowing premotoneurons, thereby inhibiting an essential motor component of feeding behavior.     

Treatment of rats with calpain inhibitors prevents sepsis-induced muscle proteolysis independent of atrogin-1/MAFbx and MuRF1 expression

Muscle wasting in sepsis is a significant clinical problem because it results in muscle weakness and fatigue that may delay ambulation and increase the risk for thromboembolic and pulmonary complications. Treatments aimed at preventing or reducing muscle wasting in sepsis, therefore, may have important clinical implications. Recent studies suggest that sepsis-induced muscle proteolysis may be initiated by calpain-dependent release of myofilaments from the sarcomere, followed by ubiquitination and degradation of the myofilaments by the 26S proteasome. In the present experiments, treatment of rats with one of the calpain inhibitors calpeptin or BN82270 inhibited protein breakdown in muscles from rats made septic by cecal ligation and puncture. The inhibition of protein breakdown was not accompanied by reduced expression of the ubiquitin ligases atrogin-1/MAFbx and MuRF1, suggesting that the ubiquitin-proteasome system is regulated independent of the calpain system in septic muscle. When incubated muscles were treated in vitro with calpain inhibitor, protein breakdown rates and calpain activity were reduced, consistent with a direct effect in skeletal muscle. Additional experiments suggested that the effects of BN82270 on muscle protein breakdown may, in part, reflect inhibited cathepsin L activity, in addition to inhibited calpain activity. When cultured myoblasts were transfected with a plasmid expressing the endogenous calpain inhibitor calpastatin, the increased protein breakdown rates in dexamethasone-treated myoblasts were reduced, supporting a role of calpain activity in atrophying muscle. The present results suggest that treatment with calpain inhibitors may prevent sepsis-induced muscle wasting.