Regulation of the guanylyl cyclase-B receptor by alternative splicing

Guanylyl cyclase-B (GC-B) is a single transmembrane receptor that binds C-type natriuretic peptide (CNP). The ligand/receptor appears critical in the regulation of cell proliferation and differentiation where it acts as an adversary of mitogenic signaling pathways. We have isolated three guanylyl cyclase-B isoforms generated from a single gene by alternative splicing and termed them GC-B1, GC-B2, and GC-B3. GC-B1 is full-length and responds maximally to CNP, GC-B2 contains a 25-amino acid deletion in the protein kinase homology domain, and GC-B3 only retains a part of the extracellular ligand-binding domain. GC-B2 binds CNP, but the ligand fails to activate the cyclase, while GC-B3 fails to bind ligand. When GC-B2 or GC-B3 is expressed coincident with GC-B1, they act as dominant negative isoforms by virtue of blocking formation of active GC-B1 homodimers. Relative expression levels of GC-B1, GC-B2, and GC-B3 vary across tissues and as a function of in vitro culture; the relative amount of GC-B2 to GC-B1 is repressed in cultured smooth muscle cells relative to endogenous ratios in the medial layer cells of the aorta. Thus, GC-B isoform levels can be independently regulated. Given that the splice variants serve as dominant negative forms, these will serve as regulators of the full-length GC-B.     

Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.     

Clusterin,an abundant serum factor,is a possible negative regulator of MT6-MMP/MMP-25 produced by neutrophils

MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.     

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.     

Oligodeoxynucleotides without CpG motifs work as adjuvant for the induction of Th2 differentiation in a sequence-independent manner

The outcomes of immune responses are regulated by various parameters including how Ags are handled by APCs. In this study, we describe the intrinsic immunomodulatory characteristics of oligodeoxynucleotides (ODNs) that improve the Ag presentation by APCs. ODNs (20-mer) containing CpG motifs induced strong Th1-skewed responses. In contrast, those without CpG motifs enhanced cytokine production by effector Th cells without particular skewing toward Th1 responses or induced the differentiation of unprimed CD4(+) T cells toward Th2 cells. These functional features were prominently envisaged when ODNs were conjugated to the Ag, and were underlain by the facilitated binding of ODN-conjugated Ag to Ia(+) cells. Despite the functional differences between ODNs with CpG motifs and those without CpG motifs, both ODNs bound to Ia(+) cells with similar affinity and kinetics. Immunoenhancing activities of the ODNs were not sequence-dependent; the characteristics, including the facilitation of Ag capture, enhancement of effector Th cell responses, and induction of Th2 cells, were shared by randomly synthesized ODNs conjugated to Ag. This is the first study suggesting that ODNs, independent of the sequences, enhance immune responses through the promoted capture of ODN-conjugated Ag by APCs.     

Foreign gene products can be enhanced by introduction into low storage protein mutants

Transgenic rice expressing soybean glycinin in its endosperm was crossed with two types of low-glutelin mutants to determine how much storage the protein mutants can contribute to increases in glycinin accumulation. The glycinin level (102 microg/100 mg seed) in the parental transgenic line was enhanced to approximately 224-237 microg/100 mg seed within a genetic background deficient in glutelin (i.e. of low glutelins). The enrichment of this foreign gene product was compensated by a decrease in the expression of other endogenous prolamine and globulin storage proteins, resulting in an almost equivalent total amount of seed storage proteins. These results show that low storage protein mutants can provide potentially useful hosts for the expression of foreign genes, allowing a higher-level accumulation, because they can provide wider space for the accumulation of foreign gene products than in the normal host plant.     

Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS. 2. Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water

This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP(8)EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP(8)EO as the sole carbon source and (18)O-labeled water as an incubation medium. In the MALDI-MS spectra of biodegraded samples, a series of OP(n)EO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OP(n)EC) were observed. The use of purified OP(8)EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates. Furthermore, the formation of OP(8)EC (the oxidized product of OP(8)EO) supported the notion that terminal oxidation is a step in the biodegradation process. When biodegradation study was carried out in (18)O-labeled water, incorporation of (18)O atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products. These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.     

Novel imidazole compounds as a new series of potent,orally active inhibitors of 5-lipoxygenase

Replacement of the dihydroquinolinone pharmacophore of Zeneca's ZD2138 by ionizable imidazolylphenyl moiety has lead to the discovery of a novel series of potent and orally active 5-lipoxygenase (5-LO) inhibitors. The synthesis and structure-activity relationship (SAR) of this series of compounds are described herein.     

A Mammalian bromodomain protein,brd4, interacts with replication factor C and inhibits progression to S phase

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.