Involvement of autocrine CXCL12/CXCR4 system in the regulation of ovarian carcinoma cell invasion

Ovarian carcinomas are often highly invasive, especially in the peritoneal cavity; however, the mechanism involved in invasion is not yet fully understood. In the present research, we studied the role of NF-κB in the invasiveness of ovarian carcinoma cells by using (−)-DHMEQ, a specific inhibitor of NF-κB. (−)-DHMEQ inhibited invasion in vitro and the expression of CXCL12 and CXCR4. We found that neutralizing antibody against CXCR4 or knockdown of CXCR4 suppressed the invasion. Proteomic analysis revealed that CXCR4-siRNA treatment lowered the secretion of several invasion-related proteins, such as MMP-9 and uPA. These data imply that (−)-DHMEQ suppressed ovarian cell invasion via inhibition of the NF-κB-regulated autocrine system of CXCL12-CXCR4.     

Ultrastructural study of spore wall morphogenesis inOphioglossum thermale kom. var.nipponicum (Miyabe et Kudo) nishida

Spore wall morphogenesis ofOphioglossum thermale var.nipponicum was examined by transmission electron microscopy. The spore wall of this species consists of three layers: endospore, exospore, and perispore. The spore wall development begins at the tetrad stage. At first, the outer undulating lamellar layer of the exospore (Lo) is formed on the spore plasma membrane in advance of the inner accumulating lamellar layer (Li) of the exospore. Next, the homogeneous layer of the exospore (H) is deposited on the outer lamellar layer. Both lamellar layers may be derived from spore cytoplasm; and the homogeneous layer, from the tapetum. Then the endospore (EN) is formed. It may be derived from spore cytoplasm. The membranous perispore (PE), derived from the tapetum, covers the exospore surface as the final layer. Though the ornamentation of this species differs distinctly from that ofO. vulgatum, the results mentioned above are fundamentally in accordance with the data obtained fromO. vulgatum (Lugardon, 1971). Therefore, the pattern of spore wall morphogenesis appears to be very stable in the genusOphioglossum.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

Tyr682 in the Aβ‐precursor protein intracellular domain regulates synaptic connectivity,cholinergic function,and cognitive performance

Processing of Aβ‐precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the ⁶⁸²YENPTY⁶⁸⁷ motif. To dissect the functions of this sequence in vivo, we created an APP knock‐in allele mutating Y⁶⁸² to Gly (APP^YG/YG mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP‐like protein 2 KO background. This evidence prompted us to characterize further the APP^YG/YG mice. Here, we show that APP^YG/YG mice develop aging‐dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP^YG/YG mice. The profound deficits caused by the Y⁶⁸² mutation underscore the biological importance of APP and indicate that APP^YG/YG are a valuable mouse model to study APP functions in physiological and pathological processes.     

Chronic delivery of antibody fragments using immunoisolated cell implants as a passive vaccination tool

Background

Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic.

Methodology and Principal Findings

We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer''s disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits.

Conclusions and Significance

The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer''s disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration.     

The Vitamin D analogue TX 527 blocks NF-kappaB activation in peripheral blood mononuclear cells of patients with Crohn's disease

Crohn's disease (CD) is an inflammatory disease characterized by the activation of the immune system in the gut. Since tumor necrosis factor (TNF-alpha) plays an important role in the initiation and perpetuation of intestinal inflammation in CD, we investigated whether TX 527 [19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)], a Vitamin D analogue, could affect peripheral blood mononuclear cells (PBMC) proliferation and exert an immunosuppressive effect on TNF-alpha production in CD patients, and whether this immunosuppressive action could be mediated by NF-kappaB down-regulation. TX 527 significantly decreased cell proliferation and TNF-alpha levels. On activation, NF-kappaB, rapidly released from its cytoplasmatic inhibitor (IKB-alpha), transmigrates into the nucleus and binds to DNA response elements in gene promoter regions. The activation of NF-kappaB, stimulated by TNF-alpha, and its nuclear translocation together with the degradation of IKB-alpha were blocked by TX 527. At the same time, NF-kappaB protein levels present in cytoplasmic extracts decreased in the presence of TNF-alpha and increased when PBMC were incubated with TX 527. The results of our studies indicate that TX 527 inhibits TNF-alpha mediated effects on PBMC and the activation of NF-kappaB and that its action is mediated by Vitamin D receptor (VDR), which is activated when the cells are stimulated with TX 527.     

Determination of topological structure of ARL6ip1 in cells: Identification of the essential binding region of ARL6ip1 for conophylline

Conophylline (CNP) has various biological activities, such as insulin production. A recent study identified ADP-ribosylation factor-like 6-interacting protein 1 (ARL6ip1) as a direct target protein of CNP. In this study, we revealed that ARL6ip1 is a three-spanning transmembrane protein and determined the CNP-binding domain of ARL6ip1 by deletion mutation analysis of ARL6ip1 with biotinyl-amino-CNP. These results suggest that CNP is expected to be useful for future investigation of ARL6ip1 function in cells. Because of the anti-apoptotic function of ARL6ip1, CNP may be an effective therapeutic drug and/or a novel chemosensitizer for human cancers and other diseases.     

Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production

Insulin stimulates production of NO in vascular endothelium via activation of phosphatidylinositol (PI) 3-kinase, Akt, and endothelial NO synthase. We hypothesized that insulin resistance may cause imbalance between endothelial vasodilators and vasoconstrictors (e.g., NO and ET-1), leading to hypertension. Twelve-week-old male spontaneously hypertensive rats (SHR) were hypertensive and insulin resistant compared with control Wistar-Kyoto (WKY) rats (systolic blood pressure 202 +/- 11 vs. 132 +/- 10 mmHg; fasting plasma insulin 5 +/- 1 vs. 0.9 +/- 0.1 ng/ml; P < 0.001). In WKY rats, insulin stimulated dose-dependent relaxation of mesenteric arteries precontracted with norepinephrine (NE) ex vivo. This depended on intact endothelium and was blocked by genistein, wortmannin, or N(omega)-nitro-l-arginine methyl ester (inhibitors of tyrosine kinase, PI3-kinase, and NO synthases, respectively). Vasodilation in response to insulin (but not ACh) was impaired by 20% in SHR (vs. WKY, P < 0.005). Preincubation of arteries with insulin significantly reduced the contractile effect of NE by 20% in WKY but not SHR rats. In SHR, the effect of insulin to reduce NE-mediated vasoconstriction became evident when insulin pretreatment was accompanied by ET-1 receptor blockade (BQ-123, BQ-788). Similar results were observed during treatment with the MEK inhibitor PD-98059. In addition, insulin-stimulated secretion of ET-1 from primary endothelial cells was significantly reduced by pretreatment of cells with PD-98059 (but not wortmannin). We conclude that insulin resistance in SHR is accompanied by endothelial dysfunction in mesenteric vessels with impaired PI3-kinase-dependent NO production and enhanced MAPK-dependent ET-1 secretion. These results may reflect pathophysiology in other vascular beds that directly contribute to elevated peripheral vascular resistance and hypertension.     

Ricerche sulla flora infestante delle colture in Italia

Summary

Studies on the weeds of cultivated land in Italy. IV: Weeds of a small farm near Montecatini Terme.

In the first part of this researche, the author gives the chief climatic data of the Montecatini Terme zone together with pedological informations about the stations offered by a small farm on which biological and statistical investigations are conducted.

All weeds (241 species) found on the area of the small farm are listed. For each species is indicated Raunhiaer's biological form, its occurence in cultivated fields or in path lanes, graves and fences.

The most of them are Therophytes (55%) and Hemicriptophytes (30%). Scarcely represented are the Geophytes (8,33%), Chamaephytes (3,75%), Phanerophytes (2,92%). Therophytes increase very much (till 78%) if only are considered the species which occur in cultivated ground.

In the second part of the work is conducted a bio-statistical study on the weeds behaviour in eight fields. Once a month, on thiese fields, statistical relieves have been made after Raunkiaer's method. The results are condensed in graphics in which, besides the percent frequences of the various weeds, are also reported the various biological stages, the types of root-system and the influence of the tillages.

Follows an examination of the weeds which have been found in the various cultivations (wheat, patatoes, corn, legumes ecc). The most important weeds (36 species) are, then, biologically and phyto-geografically classified in accordance with Korsmo's and Eig's systems, respectively. Of these weeds 81% are «seed plants» and 19% are «wandering plant» 52,7% belong to «liason group», 36,1% are polychorous or adventicious, 11,2% belong to mediterranean Element. «Stationary plants» are missing, it depends upon the fact that there are only annual crops.

At last, for the most important weeds, details are given about germination, blossoming and dissemination time, their relations with crops, their occurrence in the low or in the high part of the farm.

For a resume, conclusions are as follow:

1^°) The slope of the ground influences weed distribution on the fields of the farm; many of them (specially the winter-spring species) prefer cool fields of the low part of the farm, so that the high one is infested by a relatively small number of weeds;

2^°) the greatest number of weeds is found in the fields in spring time;

3^°) annual plants are prevailing over perennial ones;

4^°) the most infested crop is weat, the least is potatoes;

5^°) Variations of winter precipitations and, sometimes, of the sowing time may influence gravity of infestation by weeds.