Intranasal administration of an Escherichia coli-expressed codon-optimized rotavirus VP6 protein induces protection in mice

We are developing rotavirus vaccines based on the VP6 protein of the human G1P[8] [corrected] [J. Virol. 73 (1999) 7574] CJN strain of rotavirus. One prototype candidate consisting of MBP::VP6::His6, a chimeric protein of maltose-binding protein, VP6 and hexahistidine, was expressed mainly as truncated polypeptides in Escherichia coli BL21(DE3) cells. A possible reason for this extensive truncation is the high frequencies of rare bacterial codons within the rotavirus VP6 gene. Expression of truncated recombinant VP6 was found to be reduced, and expression of complete VP6 protein was simultaneously increased, when the protein was expressed in Rosetta(DE3)pLacI E. coli cells that contain increased amounts of transfer RNAs for a selection of rare codons. The same observation was made when a synthetic codon-optimized CJN-VP6 gene was expressed in E. coli BL21 or Rosetta cells. To increase protein recovery, recombinant E. coli cells were treated with 8M urea. Denatured, full-length MBP::VP6::His6 protein was then purified and used for intranasal vaccination of BALB/c mice (2 doses administered with E. coli heat-labile toxin LT(R192G) as adjuvant). Following oral challenge with the G3P[16] [corrected] [J. Virol. 76 (2002) 560] EDIM strain of murine rotavirus, protection levels against fecal rotavirus shedding were comparable (P>0.05) between groups of mice immunized with denatured codon-optimized or native (not codon-optimized) immunogen with values ranging from 87 to 99%. These protection levels were also comparable to those found after immunization with non-denatured CJN VP6. Thus, expression of complete rotavirus VP6 protein was greatly enhanced by codon optimization, and the protection elicited was not affected by denaturation of recombinant VP6.     

Immunohistochemical localization of Bis protein in the rat central nervous system

We studied the distribution of Bis (Bcl-2 interacting death suppressor) protein in the adult rat brain and spinal cord using immunohistochemistry. Immunoreactivity was observed in specific neuronal populations in distinct nuclei. The most intensely labeled cells were associated with the motor system, including most cranial nerve motor nuclei, Purkinje cells of the cerebellum, the red nucleus, and the ventral motor neurons of the spinal cord. Bis protein was also expressed in several structures associated with the ventricular system, including the subventricular zone of the lateral ventricle and its rostral extension, in the subcommissural organ, and in tanycytes, radial glial cells in the hypothalamus. Using double-labeling techniques, Bis-immunoreactive cells in the rostral migratory stream, coexpressing Bcl-2, were confirmed as glial fibrillary acidic protein-positive astrocytes comprising the glial tubes. The widespread distribution of Bis suggests that this protein has broader functions in the adult rat central nervous system than previously thought, and that it could be associated with a particular role in the rostral migratory system.J.-H. Lee and M.-Y. Lee contributed equally to this study. This work was supported by the KOSEF through the Cell Death Disease Research Center of MRC at the Catholic University of Korea (R13-2002-005-01001-0) and the Catholic Medical Center Research Foundation grant made in the program year of 2002     

Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase

Soluble guanylate cyclase (sGC) is an important downstream intracellular target of nitric oxide (NO) that is produced by endothelial NO synthase (eNOS) and inducible NO synthase (iNOS). In this study, we demonstrate that sGC exists in a complex with eNOS and heat shock protein 90 (HSP90) in aortic endothelial cells. In addition, we show that in aortic smooth muscle cells, sGC forms a complex with HSP90. Formation of the sGC/eNOS/HSP90 complex is increased in response to eNOS-activating agonists in a manner that depends on HSP90 activity. In vitro binding assays with glutathione S-transferase fusion proteins that contain the alpha- or beta-subunit of sGC show that the sGC beta-subunit interacts directly with HSP90 and indirectly with eNOS. Confocal immunofluorescent studies confirm the subcellular colocalization of sGC and HSP90 in both endothelial and smooth muscle cells. Complex formation of sGC with HSP90 facilitates responses to NO donors in cultured cells (cGMP accumulation) as well as in anesthetized rats (hypotension). These complexes likely function to stabilize sGC as well as to provide directed intracellular transfer of NO from NOS to sGC, thus preventing inactivation of NO by superoxide anion and formation of peroxynitrite, which is a toxic molecule that has been implicated in the pathology of several vascular diseases.     

A genotype-specific pollen gene associated with self-incompatibility in Lycopersicon peruvianum

Self-incompatibility is a genetically controlled process used to prevent self-pollination. We report here the characterization of pollen cDNA clones of Lycopersicon peruvianum, and the identification of a genotype-specific pollen factor involved in self-incompatibility. To identify the latter, differential mRNA display RT-PCR was performed on pollen cDNAs from S12Sa and S11Sa genotypes. We isolated four cDNA fragments expressed preferentially in S12Sa pollen, and screened a cDNA library from S12Sa pollen with the four cDNA fragments to isolate the corresponding full length cDNAs. One of the four isolated cDNAs encoded part of an actin depolymerizing factor protein that we named LpADF. LpADF is highly homologous to actin depolymerizing factors of Arabidopsis thaliana, Lilium longiflorum, and Zea mays. RNA blot analysis revealed that LpADF is only expressed in mature pollen of the S12Sa genotype, and is therefore a candidate pollen factor in the gametophyte self-incompatibility system of L. peruvianum.     

Signaling via histamine receptors in cat duodenal smooth muscle cells

Histamine produced concentration-dependent contractions in cat duodenal smooth muscle cells that were obtained by enzymatic digestion of smooth muscle with collagenase F. Pyrilamine, an H1 receptor antagonist, inhibited the contractile response while famotidine, an H2 receptor antagonist, augmented it. In cells with selectively preserved H1 receptors, produced by pretreatment with pyrilamine followed by inactivation of all unprotected receptors with N-ethylmaleimide, histamine-induced contraction was significantly augmented as compared with control cells. Pertussis toxin (PTX) had no effect on contraction, suggesting that the H1 receptor is coupled to a PTX-insensitive G protein. Gi2, Gi3, Go, Gs, and Gq subunits were present in cat duodenum, and histamine-induced contraction was inhibited by Gq antibody after cell permeabilization. Neomycin, a PLC inhibitor, inhibited the histamine-induced cell contraction, but not rhoCMB, a PLD inhibitor, or DEDA, a PLA2 inhibitor. Heparin, an IP3 receptor inhibitor, inhibited contraction whereas chelerythrine, a PKC inhibitor, had no effect. We conclude that histamine-induced contraction in cat duodenal smooth muscle cells is mediated by H1 receptors coupled to a PTX-insensitive Gq protein and results in activation of phosphatidylinositol-specific phospholipase C (PI-PLC).     

Low-dose carbon monoxide reduces airway hyperresponsiveness in mice

Carbon monoxide (CO) in expired gas has been shown to be elevated with asthma; however, its function is not known, and there is some potential that it may serve a bronchoprotective role to decrease airway hyperresponsiveness (AHR). Thus the ability of CO to reverse methacholine (MCh)-induced bronchoconstriction was evaluated in C57BL/6 (C57) and A/J mice with and without airway inflammation produced by ovalbumin (OVA). Acutely administered CO (1% in air, 10 min) reduced MCh-driven increases in lung resistance in OVA-challenged C57 mice by an average of 50% (from 14.5 to 7.1 cmH2O.ml-1.s-1), whereas no effect was observed in na?ve C57 mice or OVA-challenged C57 mice inhaling air alone. Acutely inhaled CO (500 ppm = 0.05%, for 10 min) reduced MCh-induced airway reactivity (AR) by 20-60% in airway hyperresponsive na?ve A/J mice, whereas repeated 10-min administrations of 500 ppm CO over a 5-day period decreased AR by 50%. Repeated administration of low-dose CO [250 (0.025%) and (0.05%) 500 ppm, 1 h/day, 5 days] to A/J mice with airway inflammation likewise resulted in a drop of AR by 50%, compared with those not receiving CO. Inhibition of guanylyl cyclase/guanosine 3',5'-cyclic monophosphothioate (cGMP) using 1H-[1,2,4] oxydiazolo[4,3-a]quinoxalin-1-one or a competitive inhibitor, Rp diastereomers of 8-bromo-cGMP, resulted in inhibition of the effect of CO on AHR, suggesting that the effects of CO were mediated through this mechanism. These results indicate that low-dose CO can effectively reverse AHR in the presence and absence of airway inflammation in mice and suggest a potential role for CO in the modulation of AHR.     

Alterations and reversibility in the chromatin,cytoskeleton and development of pig oocytes treated with roscovitine

Germinal vesicle (GV) breakdown in mammalian oocytes is regulated by the activation of maturation promoting factor (MPF). We investigated a specific cdc2 kinase inhibitor, roscovitine, to maintain pig oocytes in the GV stage. Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse ovaries and cultured for 44 hr in NCSU#23 medium containing different levels of roscovitine (0, 10, 20, 30, 40, 50 microM in Experiment 1 and 0, 40, 60, 80, 100, 120 microM in Experiment 2). The COCs were cultured for another 44 hr after removal of the chemical. Twenty oocytes in each group were fixed at 44 hr for immunocytochemical labeling of the cytoskeleton and the rest (approximately 20/group) were fixed at the end of 88 hr after culture. Results showed that the inhibition of the oocyte in the GV stage was not effective when 10-50 microM (Experiment 1) of roscovitine were used (19-34%). When oocytes were released from the inhibitor, similar proportions (70-83%) of oocytes were observed in the MII or advanced stages among treatments. However, when higher concentrations of roscovitine were used (Experiment 2), significantly greater inhibitory effect was observed at the levels of 80-120 microM with 83-91% oocytes being blocked in the GV stage when compared to the control (9%) and the 40-60 microM (27-43%) groups (P < 0.05). Although 15-21% of the oocytes showed abnormal MII morphology with aberrant meiotic spindles and/or formation of cytoplasmic microtubules, a substantial number of oocytes resumed meiosis and reached MII stage at 44 hr after removal of this chemical. In Experiment 3, different concentrations of roscovitine (0, 20, 40, and 80 microM) were tested to examine the length of intervals (0, 11, 22, 33, and 44 hr) for an effective inhibition. Results showed that the inhibitory effect was significantly more prominent at 22 hr than that at 33 and 44 hr after roscovitine treatment in all treatment groups (P < 0.05). This study demonstrated that roscovitine-treated oocytes resumed meiosis after removal of the inhibitor. This could provide flexibility for studying porcine oocyte development and embryo cloning and may have application in other species.