MicroRNA-31 Is Overexpressed in Cutaneous Squamous Cell Carcinoma and Regulates Cell Motility and Colony Formation Ability of Tumor Cells

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.     

Evolutionary implications of a complex pattern of DNA sequence homology extending far upstream of the hsp70 genes at loci 87A7 and 87C1 in Drosophila melanogaster

Genes coding for the major 70,000 M_r heat shock protein (hsp70) are found at two loci, 87A7 and 87C1, in Drosophila melanogaster. At 87A7 they are present as two genes in diverging orientation, whilst at 87C1 two tandemly repeated distal copies are separated from a single copy in divergent orientation by about 40,000 bases of DNA. Within this 40,000 bases are found the αβ heat-induced genes, interspersed with γ elements. In this paper we report the isolation and characterization of the proximal hsp70 gene from locus 87C1. The DNA sequence upstream from this gene shows greater than 98% homology with that of αγ, suggesting that the γ element interspersed with αβ sequences originated from this position. In addition, we present the DNA sequence between the two genes in a cloned DNA segment from 87A7, and compare the sequence with those from 87C1. We find a complex pattern of nucleotide sequence homology extending far upstream of the hsp70 genes at the two loci. The evolution of the present arrangement at these two loci is discussed.     

Effects of orexin-monoaminergic interactions on oxytocin secretion in rat neurohypophyseal cell cultures

The effects of orexin-monoaminergic compound interactions on oxytocin release were studied in 14-day rat neurohypophyseal cell cultures prepared by an enzymatic dissociation technique. The oxytocin contents of the supernatants were determined by radioimmunoassay. Following the administration of orexin-A or orexin-B in increasing doses, significant changes were not observed in the oxytocin content of the supernatant media. The oxytocin level increased substantially in response to adrenaline, noradrenaline, serotonin, histamine, dopamine or K(+) treatment. Preincubation with orexin-A or orexin-B reduced the adrenaline-, histamine- or serotonin-induced oxytocin level increases, but the oxytocin concentrations of the supernatant media remained above the control level. There was no significant difference in decreasing effect between orexin-A and orexin-B. Neither orexin-A nor orexin-B induced changes in oxytocin release following monoaminergic compound treatment. The results indicate that the changes in oxytocin secretion induced by the monoaminergic system can be directly influenced by the orexin system. The effects of orexin on oxytocin release can be antagonized by an orexin-1 receptor-specific antagonist. It may be presumed that the orexins can play a role in the pathogenetic process of metabolic diseases (e.g. obesity) by reducing the effects of increased oxytocin release caused by monoaminergic compounds. The interactions between the monoaminergic and orexin systems regarding oxytocin secretion occur at both the hypothalamic and the neurohypophyseal levels.     

Topoisomerase II cleavage in chromatin

We have examined the effect of the anti-tumor drug VM-26 on purified Drosophila topoisomerase II, and used this drug to map (putative) topoisomerase II cleavage sites in chromatin. These studies indicate that VM-26 interferes with the strand breakage-rejoining catalytic cycle. VM-26 appears to stabilize the topoisomerase-II-cleavable complex and markedly enhances the formation of double-strand breaks in naked DNA. VM-26 also stimulates the formation of double-strand breaks in isolated Drosophila nuclei. Analysis of the parameters of the VM-26-stimulated cleavage reaction in nuclei strongly suggests that the double-strand scissions are generated by endogenous topoisomerase II. Finally, we have examined the distribution of (putative) cleavage sites for endogenous topoisomerase II in the chromatin of the 87A7 heat shock locus and the histone repeat unit. We have found that there are prominent VM-26-induced cleavage products from the 5' ends of the 87A7, the two heat shock protein 70 genes, and in the intergenic spacer separating these genes. Moreover, the pattern of VM-26-induced cleavage products is altered in nuclei prepared from heat-shocked cells. In the case of the histone repeat unit, only minor VM-26-induced cleavage products are observed in nuclei (in spite of the fact that experiments on naked DNA indicate that the histone repeat contains many major cleavage sites for purified topoisomerase II). These findings suggest that the nucleoprotein organization of different DNA segments may be important in determining whether specific sites are accessible to endogenous topoisomerase II in nuclei.     

26S proteasome subunits are O-linked N-acetylglucosamine-modified in Drosophila melanogaster

The O-linked glycosylation of highly purified Drosophila 26S proteasome has been analyzed by immunological and lectin-binding methods. Five regulatory complex subunits and at least nine catalytic core subunits were recognized by two different monoclonal antibodies specific for O-linked N-acetylglucosamine-modified proteins, and by wheat germ agglutinin, which is specific for the N-acetylglucosamine sugar side-chain. The specificity of these reactions has been proved by competition studies with free N-acetylglucosamine. Three ATPase subunits of the regulatory complex, which are O-glycosylated, have previously been shown [FEBS Lett. 430 (1998) 269] to occur in phosphorylated form as well, indicating that several different post-translational modifications, with distinct regulatory potential, may be present on the same subunit.     

Increased intracellular calcium level and impaired nutrient absorption are important pathogenicity traits in the chicken intestinal epithelium during <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> colonization

Although a high number of chickens carry Campylobacter jejuni, the mechanistic action of colonization in the intestine is still poorly understood. The current study was therefore designed to investigate the effects of C. jejuni on glucose uptake, amino acids availability in digesta, and intracellular calcium [Ca²⁺]_i signaling in the intestines of broiler chickens. For this, we compared: control birds (n?=?60) and C. jejuni-infected birds (n?=?60; infected orally with 1?×?10⁸ CFU of C. jejuni NCTC 12744 at 14 days of age). Our results showed that glucose uptake was reduced due to C. jejuni infection in isolated jejunal, but not in cecal mucosa at 14 days postinfection (dpi). The decrease in intestinal glucose absorption coincided with a decrease in body weight gain during the 2-week post-infectious period. A reduction in the amount of the amino acids (serine, proline, valine, leucine, phenylalanine, arginine, histidine, and lysine) in ileal digesta of the infected birds at 2 and/or 7 dpi was found, indicating that Campylobacter utilizes amino acids as a carbon source for their multiplication. Applying the cell-permeable Ca²⁺ indicator Fluo-4 and two-photon microscopy, we revealed that [Ca²⁺]_i was increased in the jejunal and cecal mucosa of infected birds. The muscarinic agonist carbachol induced an increase in [Ca²⁺]_i in jejunum and cecum mucosa of control chickens, a response absent in the mucosa of infected chickens, demonstrating that the modulation of [Ca²⁺]_i by Campylobacter might be involved in facilitating the necessary cytoskeletal rearrangements that occur during the bacterial invasion of epithelial cells. In conclusion, this study demonstrates the multifaceted interactions of C. jejuni with the gastrointestinal mucosa of broiler chickens. For the first time, it could be shown that a Campylobacter infection could interfere with intracellular Ca²⁺ signaling and nutrient absorption in the small intestine with consequences on intestinal function, performance, and Campylobacter colonization. Altogether, these findings indicate that Campylobacter is not entirely a commensal and can be recognized as an important factor contributing to an impaired chicken gut health.     

Role of the Deubiquitylating Enzyme DmUsp5 in Coupling Ubiquitin Equilibrium to Development and Apoptosis in Drosophila melanogaster

Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent.     

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.