In situ localization of TNFalpha/beta,TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue

Tumor necrosis factor (TNF) has been implicated in several infectious and inflammatory lung diseases. Two closely related variants, TNFalpha and TNFbeta, elicit various cellular responses via two distinct TNF receptors, the 55-kDa TNF-R1 and the 75-kDa TNF-R2. Recently, a TNFalpha-converting enzyme (TACE) was described, which cleaves and releases the membrane-bound TNFalpha. In the present study in normal rat and human lung tissue, the constitutive expression of TNFalpha/beta, TACE and TNF-R1/R2 was investigated by immunohistochemical techniques. In addition, TNFalpha and TNFbeta mRNA were localized by in situ hybridization. Both TNFalpha and TNFbeta were detected in various lung cell types. Expression of TNFalpha was particularly prominent in bronchial epithelial cells and vascular smooth muscle cells, next to alveolar macrophages. Both in situ hybridization for TNFalpha message and TACE immunostaining matched this expression profile. TNFbeta-so far only known to be produced by lymphocytes-was demonstrated in alveolar macrophages, bronchial epithelial cells, vascular smooth muscle cells and endothelial cells at the protein and the message level. Both TNF receptors were detected, with TNF-R1 being prominent on bronchial epithelial cells and endothelial cells, and TNF-R2 being expressed by nearly all cell types. Following LPS stimulation in isolated rat lungs TNFalpha/beta signal intensity was largely reduced due to liberation of stored TNFalpha/beta, while TACE immunoreactivity remained unchanged or was enhanced, demonstrating increased TNF generation.We conclude that both TNFalpha and TNFbeta are constitutively expressed by several non-leukocytic cell types in the human and rat lung. In concert with the expression of TACE and the TNF receptors R1 and R2, this finding suggests in addition to the known role of the TNF system in inflammation physiological functions of the TNF system in different compartments of the adult lung, with the vasculature and the bronchial tissue being of particular interest in addition to the leukocyte/macrophage populations.     

Lysophosphatidic acid triggers cathepsin B-mediated invasiveness of human endometriotic cells

Extracellular lysophosphatidic acid (LPA) and the G-protein-coupled LPA receptors (LPAR) are involved in cell migration and invasion and found in the human endometrium. However, underlying mechanisms resulting in cellular invasion have been rarely investigated. We used stromal endometrial T-HESC, epithelial endometriotic 12Z, 49Z and Ishikawa cells. Interestingly, proliferation of T-HESC cells was strongly increased after LPA treatment, whereas the epithelial cell lines only showed a moderate increase. LPA increased invasion of 12Z and 49Z strongly and significantly. The LPAR inhibitor Ki16425 (LPAR1/3) attenuated significantly LPA-induced invasiveness of 12Z, which was confirmed by LPAR1 and LPAR3 siRNAs, showing that both LPA receptors contribute to invasiveness of 12Z cells. Investigation of cell invasion with an antibody-based protease array revealed mainly differences in cathepsins and especially cathepsin B between 12Z compared to the less invasive Ishikawa. Stimulation with LPA showed a time- and dose-dependent increased secretion of cathepsin B which was inhibited by the Gq inhibitor YM-254890 and Gi/o inhibitor pertussis toxin in the 12Z cells, again highlighting the importance of LPAR1/3. The activity of intracellular and secreted cathepsin B was significantly upregulated in LPA-treated samples. Inhibition of cathepsin B with the specific inhibitor CA074 significantly reduced LPA-increased invasion of 12Z. Our results reveal a novel role of LPA-mediated secretion of cathepsin B which stimulated invasion of endometriotic epithelial cells mainly via LPAR1 and LPAR3. These findings may deepen our understanding how endometriotic cells invade into ectopic sites, and provide new insights into the role of LPA and cathepsin B in cellular invasion.     

Identification of a set of RFLP probes for subspecies differentiation in Oryza sativa L.

Sixty-eight indica-japonica tester-differentiating RFLP probes were tested in seven indica and seven japonica varieties of rice (Oryza sativa L.) with four enzyme digestions (EcoRI, EcoRV, HindIII and DraI). Twenty-one DNA clones were isolated as indica-japonica subspecies-differentiating probes. A set of 13 probes was established as core probes for subspecies differentiation and a pooled blotting analysis was carried out to facilitate the application of RFLP in rice genetics and breeding practice. A dendrogram of 12 wide-compatibility varieties was constructed based on RFLPs detected by 13 core probes with single enzyme digestions. It was speculated that most RFLPs of indica-japonica differentiating probes were generated by insertions/deletions, which may be of great significance for the origin and differentiation of subspecies in Oryza sativa L.     

Testing for clonal propagation

The conceptual basis for testing clonal propagation is reconsidered with the result that two steps need to be distinguished clearly: (1) specification of the characteristics of multilocus genotype frequencies that result from sexual reproduction together with the kinds of deviations from these characteristics that are produced by clonal propagation, and (2) a statistical method for detecting these deviations in random samples. It is pointed out that a meaningful characterization of sexual reproduction reflects the association of genes in (multilocus) genotypes within the bounds set by the underlying gene frequencies. An appropriate measure of relative gene association is developed which is equivalent to a multilocus generalization of the standardized gametic disequilibrium (linkage disequilibrium). Its application to the characterization of sexually produced multilocus genotypes is demonstrated. The resulting hypothesis on the frequency of a sexually produced genotype is tested with the help of the (significance) probability of obtaining at least two copies of the genotype in question in a random sample of a given size. If at least two copies of the genotype are observed in a sample, and if the probability is significant, then the hypothesis of sexual reproduction is rejected in favor of the assumption that all copies of the genotype belong to the same clone. Common testing approaches rest on the hypothesis of completely independent association of genes in genotypes and on the (significance) probability of obtaining at least as many copies of a genotype as observed in a sample. The validity of these approaches is discussed in relation to the above considerations and recommendations are set out for conducting appropriate tests.     

Cancer of the endometrium: current aspects of diagnostics and treatment

Background  

Endometrial cancer represents a tumor entity with a great variation in its incidence throughout the world (range 1 to 25). This suggests enormous possibilities of cancer prevention due to the fact that the incidence is very much endocrine-related, chiefly with obesity, and thus most frequent in the developed world. As far as treatment is concerned, it is generally accepted that surgery represents the first choice of treatment. However, several recommendations seem reasonable especially with lymphadenectomy, even though they are not based on evidence. All high-risk cases are generally recommended for radiotherapy.     

Real-time closed-loop electrophysiology: towards new frontiers in in vitro investigations in the neurosciences

Reflected at any level of organization of the central nervous system, most of the processes ranging from ion channels to neuronal networks occur in a closed loop, where the input to the system depends on its output. In contrast, most in vitro preparations and experimental protocols operate autonomously, and do not depend on the output of the studied system. Thanks to the progress in digital signal processing and real-time computing, it is now possible to artificially close the loop and investigate biophysical processes and mechanisms under increased realism. In this contribution, we review some of the most relevant examples of a new trend in in vitro electrophysiology, ranging from the use of dynamic-clamp to multi-electrode distributed feedback stimulation. We are convinced these represents the beginning of new frontiers for the in vitro investigation of the brain, promising to open the still existing borders between theoretical and experimental approaches while taking advantage of cutting edge technologies.     

The novel miR-9500 regulates the proliferation and migration of human lung cancer cells by targeting Akt1

MicroRNAs have crucial roles in lung cancer cell development. They regulate cell growth, proliferation and migration by mediating the expression of tumor suppressor genes and oncogenes. We identified and characterized the novel miR-9500 in human lung cancer cells. The miR-9500 forms a stem-loop structure and is conserved in other mammals. The expression levels of miR-9500 were reduced in lung cancer cells and lung cancer tissues compared with normal tissues, as verified by TaqMan miRNA assays. It was confirmed that the putative target gene, Akt1, was directly suppressed by miR-9500, as demonstrated by a luciferase reporter assay. The miR-9500 significantly repressed the protein expression levels of Akt1, as demonstrated via western blot, but did not affect the corresponding mRNA levels. Akt1 has an important role in lung carcinogenesis, and depletion of Akt1 has been shown to have antiproliferative and anti-migratory effects in previous studies. In the current study, the overexpression of miR-9500 inhibited cell proliferation and the expression of cell cycle-related proteins. Likewise, the overexpression of miR-9500 impeded cell migration in human lung cancer cells. In an in vivo assay, miR-9500 significantly suppressed Fluc expression compared with NC and ASO-miR-9500, suggesting that cell proliferation was inhibited in nude mice. Likewise, miR-9500 repressed tumorigenesis and metastasis by targeting Akt1. These data indicate that miR-9500 might be applicable for lung cancer therapy.MicroRNAs (miRNAs) are small, non-coding RNAs, 18–25 nucleotides (nt) in length that regulate gene expression by binding to the 3′-untranslated region (UTR) of their target genes,^{1, 2} and these RNAs are processed from introns, exons or intergenic regions.³ First, miRNAs are transcribed by RNA polymerase II into primary miRNA (pri-miRNA) molecules that contain several thousand nucleotides. The pri-miRNAs are then sequentially processed by a microprocessor, such as Drosha RNase III endonuclease and DiGeorge syndrome region gene 8 protein (DGCR8), to form ∼70 nt-stem-loop intermediates known as miRNA precursors (pre-miRNAs).^{4, 5} The pre-miRNAs are then exported from the nucleus into the cytoplasm via Exportin-5 (EXP5), with its cofactor Ran-GTP; in the cytoplasm, these pre-miRNAs are processed into 18–25 nt mature miRNA duplexes by the RNase III endonuclease Dicer.^{6, 7} The mature miRNA duplexes, along with the Argonaute proteins, are integrated as single-stranded RNAs into an RNA-induced silencing complex, which induces either the cleavage or the translational inhibition of the targeted mRNAs.^{8, 9, 10} miRNAs have been implicated in a variety of biological processes associated with cancer development, including cell proliferation and invasion,¹¹ and miRNA expression is deregulated in many forms of cancer.¹²Cancer is a major public health problem worldwide. Lung cancer represents one of the most predominant types of cancer, with high mortality rates in both men and women. Epithelial lung cancer can be categorized into one of two types: small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC accounts for ∼80% of lung cancer cases, and these cases can be further categorized as adenocarcinoma (40%), squamous cell carcinoma (30–35%), and large cell carcinoma (5–15%). NSCLC has a 5-year survival rate of only 16%.^{13, 14, 15} Current studies have shown that miRNAs are deregulated in various cancers, including NSCLC, and may act as oncogenes or tumor suppressor genes.¹⁶ For example, the Let-7 family,¹⁷ miR-15a/16,¹⁸ miR-17-92,¹⁹ miR-107 and miR-185,²⁰ are deregulated in lung cancer.Some studies have reported that phosphatidylinositol 3-kinase (PI3K) signaling is activated in human cancers^{21, 22} and has an important role in the progression of NSCLC. The PI3K pathway modulates several cellular mechanisms, such as cell survival, proliferation, migration and motility, and thereby significantly affects the growth of tumors.^{23, 24} The primary regulator of the PI3K pathway is Akt, a protein kinase B that mediates cell survival, cell death,²⁵ cell growth, cell migration and angiogenesis.^{26, 27, 28} The silencing of the Akt1 gene has been shown to inhibit the proliferation of gastric cancer cells both in vitro and in vivo.²⁹ Other studies have shown that aberrant AKT activation has a critical role in tumorigenesis.³⁰In this study, we identified small RNAs in lung cancer cells. To analyze a novel miRNA signature, we examined the structure and sequence of the small RNAs, analyzed the expression patterns of the novel miRNAs in lung cancer tissues and assessed the miRNA target genes. Our data revealed that miR-9500 regulates certain human lung cancer cell functions, including cell growth, proliferation, and migration.     

Effect of the tropical tannin-rich shrub legumes Calliandra calothyrsus and Flemingia macrophylla on methane emission and nitrogen and energy balance in growing lambs

The objective of this study was to test whether the use of tannin-rich shrub legume forage is advantageous for methane mitigation and metabolic protein supply at unchanged energy supply when supplemented in combination with tannin-free legumes to sheep. In a 6 × 6 Latin-square design, foliage of two tannin-rich shrub legume species (Calliandra calothyrsus and Flemingia macrophylla) were used to replace either 1/3 or 2/3, respectively, of a herbaceous high-quality legume (Vigna unguiculata) in a diet composed of the tropical grass Brachiaria brizantha and Vigna in a ratio of 0.55 : 0.45. A Brachiaria-only diet served as the negative control. Each experimental period lasted for 28 days, with week 3 serving for balance measurement and data collection inclusive of a 2-day stay of the sheep in open-circuit respiration chambers for measurement of gaseous exchange. While Vigna supplementation improved protein and energy utilisation, the response to the partial replacement with tannin-rich legumes was less clear. The apparent total tract digestibilities of organic matter, NDF and ADF were reduced when the tannin-rich plants partially replaced Vigna, and the dose-response relationships were mainly linear. The tannin-rich plants caused the expected redistribution of more faecal N in relation to urinary N. While Flemingia addition still led to a net body N retention, even when fed at the higher proportion, adding higher amounts of Calliandra resulted in body protein mobilisation in the growing lambs. With respect to energy, supplementation of Vigna alone improved utilisation, while this effect was absent when a tannin-rich plant was added. The inclusion of the tannin-rich plants reduced methane emission per day and per unit of feed and energy intake by up to 24% relative to the Vigna-only-supplemented diet, but this seems to have been mostly the result of a reduced organic matter and fibre digestion. In conclusion, Calliandra seems less apt as protein supplement for ruminants while Flemingia could partially replace a high-quality legume in tropical livestock systems. However, methane mitigation would be small due to associated reductions in N and energy retention.