ER stress response induced by the production of human IL-7 in rice endosperm cells

Rice seed has been used as a production platform for high value recombinant proteins. When mature human interleukin 7 (hIL-7) was expressed as a secretory protein in rice endosperm by ligating the N terminal glutelin signal peptide and the C terminal KDEL endoplasmic reticulum (ER) retention signal to the hIL-7 cytokine to improve production yield, this protein accumulated at levels visible by Coomassie Brilliant Blue staining. However, the production of this protein led not only to a severe reduction of endogenous seed storage proteins but also to a deterioration in grain quality. The appearance of aberrant grain phenotypes (such as floury and shrunken) was attributed to ER stress induced by the retention of highly aggregated unfolded hIL-7 in the ER lumen, and the expression levels of chaperones such as BiPs and PDIs were enhanced in parallel with the increase in hIL-7 levels. The activation of this ER stress response was shown to be mainly mediated by the OsIRE1-OsbZIP50 signal cascade, based on the appearance of unconventional splicing of OsbZIP50 mRNA and the induction of OsBiP4&5. Interestingly, the ER stress response could be induced by lower concentrations of hIL-7 versus other types of cytokines such as IL-1b, IL-4, IL-10, and IL-18. Furthermore, several ubiquitin 26S proteasome-related genes implicated in ER-associated degradation were upregulated by hIL-7 production. These results suggest that severe detrimental effects on grain properties were caused by proteo-toxicity induced by unfolded hIL-7 aggregates in the ER, resulting in the triggering of ER stress.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

RECQL1 DNA Repair Helicase: A Potential Therapeutic Target and a Proliferative Marker against Ovarian Cancer

Objective

This study analyzed the clinicopathological correlation between ovarian cancer (OC) and RECQL1 DNA helicase to assess its therapeutic potential.

Methods

Surgically resected OC from 118 retrospective cases, for which paraffin blocks and all clinical data were complete, were used in this study. RECQL1 and Ki-67 immunostaining were performed on sections to correlate RECQL1 staining with subtype and patient survival. Ten OC and two normal cell lines were then examined for RECQL1 expression and were treated with siRNA against RECQL1 to assess its effect on cell proliferation.

Results

Of the 118 cases of adenocarcinoma (50, serous; 26, endometrioid; 21, clear cell; 15, mucinous; 6, other histology), 104 (90%) showed varying levels of RECQL1 expression in the nuclei of OC cells. The Cox hazards model confirmed that diffuse and strong staining of RECQL1 was correlated with histological type. However, RECQL1 expression did not correlate with overall patient survival or FIGO stage. In vitro, RECQL1 expression was exceptionally high in rapidly growing OC cell lines, as compared with normal cells. Using a time-course analysis of RECQL1-siRNA transfection, we observed a significant inhibition in cell proliferation.

Conclusions

RECQL1 DNA helicase is a marker of highly proliferative cells. RECQL1-siRNA may offer a new therapeutic strategy against various subtypes of OC, including platinum-resistant cancers, or in recurrent cancers that gain platinum resistance.     

Antihypertensive activity of transgenic rice seed containing an 18-repeat novokinin peptide localized in the nucleolus of endosperm cells

Novokinin (Arg-Pro-Leu-Lys-Pro-Trp, RPLKPW) is a new potent antihypertensive peptide based on the sequence of ovokinin (2-7) derived from ovalbumin. We previously generated transgenic rice seeds in which eight novokinin were fused to storage protein glutelins (GluA2 and GluC) for expression. Oral administration of these seeds to spontaneously hypertensive rats (SHRs) reduced systolic blood pressures at a dose of 1 g seed/kg of SHR. Here, 10- or 18-tandem repeats of novokinin with an endoplasmic reticulum (ER) retention signal (Lys-Asp-Glu-Leu, KDEL) at the C terminus were directly expressed in rice under the control of the glutelin promoter containing its signal peptide. Only small amounts of the 18-repeat novokinin accumulated, and it was unexpectedly deposited in the nucleolus. This abnormal intracellular localization was explained by an endogenous signal for nuclear localization. The GFP reporter protein fused to this sequence targeted to nuclei by a transient assay using onion epidermal cells. Transgenic seed expressing the 18-repeat novokinin exhibited significantly higher antihypertensive activity after a single oral dose to SHR even at one-quarter the amount (0.25 g/kg) of the transgenic rice seed expressing the fusion construct; though, its novokinin content was much lower (1/5). Furthermore, in a long-term administration for 5 weeks, even a smaller dose (0.0625 g/kg) of transgenic seeds could confer antihypertensive activity. This high antihypertensive activity may be attributed to differences in digestibility of expressed products by gastrointestinal enzymes and the unique intracellular localization. These results indicate that accumulation of novokinin as a tandemly repeated structure in transgenic rice is more effective than as a fusion-type structure.     

Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.     

Ground-surface application of pheromones through a mini-dispenser for mating disruption of the white grub beetle <Emphasis Type="Italic">Dasylepida ishigakiensis</Emphasis> (Coleoptera: Scarabaeidae)

The effect of mating disruption by the ground-surface application of 2-cm-long dispensers (mini-dispensers) of 2-butanol against the white grub beetle Dasylepida ishigakiensis Niijima et Kinoshita (Coleoptera: Scarabaeidae) was examined in sugarcane fields on the Miyako Island, Okinawa, Japan. Mating rates and male catches with sex pheromone traps were reduced to a low level comparable to that obtained from a conventional method in which rope dispensers (25 m) were hung at a height of ca. 30 cm along the sugarcane ridges. Both mating rates and male catches were reduced with increasing number of treated mini-dispensers. These results suggest that by using mini-dispensers the amounts of synthetic sex pheromone and plastic resin can be reduced to 1/10–1/5 and 1/6–1/3, respectively, of their conventional application with rope-type dispensers, without impairing the efficiency of mating disruption in this beetle. Furthermore, the amount of labor required for the application of this method is expected to be greatly reduced compared to the rope-type dispenser method.     

Molecular characterization of a novel family-46 chitosanase from Pseudomonas sp. A-01

Pseudomonas sp. A-01, isolated as a strain with chitosan-degrading activity, produced a 28 kDa chitosanase. Following purification of the chitosanase (Cto1) and determination of its N-terminal amino acid sequence, the corresponding gene (cto1) was cloned by a reverse-genetic technique. The gene encoded a protein, composed of 266 amino acids, including a putative signal sequence (1-28), that showed an amino acid sequence similar to known family-46 chitosanases. Cto1 was successfully overproduced and was secreted by a Brevibacillus choshinensis transformant carrying the cto1 gene on expression plasmid vector pNCMO2. The purified recombinant Cto1 protein was stable at pH 5-8 and showed the best chitosan-hydrolyzing activity at pH 5. Replacement of two acidic amino acid residues, Glu23 and Asp41, which correspond to previously identified active centers in Streptomyces sp. N174 chitosanase, with Gln and Asn respectively caused a defect in the hydrolyzing activity of the enzyme.     

Inhibition of fibronectin binding of some bacterial cells by subtle pH increase within the physiological range

The fibronectin (Fn)-binding ability of microorganisms is considered to be involved in their pathogenicities. Granulicatella adiacens, a member of the oral flora and a causative agent of culture-negative infective endocarditis, showed nearly maximum binding to immobilized Fn at pH 7.2 but greatly reduced binding at a slightly higher pH 7.4 and almost no binding at pH 7.6 in the presence of physiological concentration of NaCl (0.15 M). A similar pH-sensitive Fn-binding property was noted with Escherichia coli and Abiotrophia defectiva, but not with Streptococcus pyogenes nor Staphylococcus aureus. In contrast, bindings to laminin and fibrinogen observed for some of these strains were unaffected by the same pH changes. This fastidious pH-dependency of Fn-binding abilities of some bacteria warns that the pH condition must be seriously considered in the in vitro assay of bacterial adherence to fibronectin.