Roles of IκB kinase ε in the innate immune defense and beyond

IκB kinase ε(IKKε) is a non-canonical IκB kinase that is extensively studied in the context of innate immune response. Recently, significant progress has been made in understanding the role of IKKεin interferon(IFN) signaling. In addition to its roles in innate immunity, recent studies also demonstrate that IKKε is a key regulator of the adaptive immune response. Specifically, IKKεfunctions as a negative feedback kinase to curtail CD8 T cell response, implying that it can be a potential therapeutic target to boost antiviral and antitumor T cell immunity. In this review, we highlight the roles of IKKε in regulating IFN signaling and T cell immunity, and discuss a few imminent questions that remain to be answered.     

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.     

The assessing of Pacific oyster (Crassostrea gigas) rearing performances by the IFREMER/REMORA network: method and first results (1993–98) in Brittany (France)

Parasitological monitoring was carried out from 1997 to 1999 in a highly saline (41–45 g/l) lake in southeastern California, Salton Sea. A total of 1473 fishes were examined. Young tilapia, Oreochromis mossambicus, croaker, Bairdiella icistia, and mudsucker, Gillichthys mirabilis, were found infected by ectoparasites. Some persistent foci of fish infestations were found around the perimeter of the lake. The diversity of parasites was limited to three protozoan species, Amyloodinium ocellatum (Dinoflagellida), Ambiphrya ameiuri (Peritricha), Cryptobia branchialis (Bodonida: Kinetoplastida), and two metazoans, the monogeneans Gyrodactylus olsoni and G. imperialis. Both A. ocellatum and A. ameiuri infested fish from spring through fall. The greatest infestations occurred in summer (29–40°C) in the case of A. ocellatum and in spring and autumn (22–27°C) in the case of A. ameiuri. High parasite loads caused severe damage to such respiratory organs as gills and skin. They may depress respiration and osmoregulation and, in combination with other environmental factors, cause fish suffocation and death. These parasites may play a major role in juvenile fish mortality.     

Perkinsus marinus in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of México

Culture of the pleasure oyster Crassostrea corteziensis is emerging as an alternative to the Pacific oyster (Crassostrea gigas) for oyster producers, who face severe mortalities since 1997 in Northwest México. For determining the health status of this species, we conducted a histopathological analysis of cultured populations from two estuaries in the Pacific coast of México. Macroscopical analysis revealed animals with transparent and retracted mantle. Histopathological analysis of these specimens showed tissue alterations and parasitic forms consistent with Perkinsus sp. infection. Stages of the parasite identified included tomont and trophozoites with an eccentric vacuole characteristic of Perkinsus spp. Pieces of tissues of infected oysters were incubated in Fluid Thioglycollate Medium (FTM) resulting in blue–black hypnospores after incubation. The identity of the parasite was confirmed by species specific PCR-based assay in DNA samples from oysters, tissue fractions from FTM cultures, and deparaffined samples with Perkinsus-like parasite detected by histology. Sequencing of positive amplified fragments (307 bp) showed a sequence similar to Perkinsus marinus strain TXsc NTS ribosomal RNA gene (100% coverage and 98% identity, GenBank Accession No. AF497479.1) and to P. marinus, Genomic DNA, (100% coverage and 97% identity, GenBank Accession No. S78416.1). The prevalence of P. marinus varied from 1 to 5% in Boca del Camichín and from 1 to 6% in Pozo Chino. In general, the intensity of infection was moderate. The infection was observed in oysters from 31 to 110 mm of shell length. This is the first record of P. marinus in oysters from the North America Pacific coast and the first record in C. corteziensis. The origin of this parasite in the area is unknown, but it may be associated to introductions of Crassostrea virginica from the East coast of United States of America or Gulf of México.     

In Vivo RNA Interference of a Gonad-Specific Transforming Growth Factor-β in the Pacific Oyster Crassostrea gigas

We investigated the role of oyster gonadal TGFβ (og-TGFβ) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFβ, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFβ dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFβ appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.     

Introduced Pacific oysters (Crassostrea gigas) in the northern Wadden Sea: invasion accelerated by warm summers?

Among the increasing number of species introduced to coastal regions by man, only a few are able to establish themselves and spread in their new environments. We will show that the Pacific oyster (Crassostrea gigas) took 17 years before a large population of several million oysters became established on natural mussel beds in the vicinity of an oyster farm near the island of Sylt (northern Wadden Sea, eastern North Sea). The first oyster, which had dispersed as a larva and settled on a mussel bed, was discovered 5 years after oyster farming had commenced. Data on abundance and size-frequency distribution of oysters on intertidal mussel beds around the island indicate that recruitment was patchy and occurred only in 6 out of 18 years. Significant proportions of these cohorts survived for at least 5 years. The population slowly expanded its range from intertidal to subtidal locations as well as from Sylt north- and southwards along the coastline. Abundances of more than 300 oysters m^–2 on mussel beds were observed in 2003, only after two consecutive spatfalls in 2001 and 2002. Analyses of mean monthly water temperatures indicate that recruitment coincided with above-average temperatures in July and August when spawning and planktonic dispersal occurs. We conclude that the further invasion of C. gigas in the northern Wadden Sea will depend on high late-summer water temperatures.Communicated by H.D. Franke     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

Gonadic cycle of the American oyster Crassostrea virginica (Lamellibranchia: Ostreidae) in Mecoacán, Tabasco, México

The American oyster Crassostrea virginica is exploited along Gulf of Mexico. This resource represents a job source and incomes for fishermen. In Mexico the production is supported by Tabasco state, the first producer. However, the mexican landings of this bivalve had been dropped about 40% last ten years. By 1999, Tabasco presents a unique ban season fishery of oysters. This season was based in evaluation of gonadal development by visual observations of color and texture, larvae and seeds abundance. In 2000, the government set up two ban seasons in terms of evaluation of populations every year, without gonadal analysis. In general, exists different spawning seasons for American oyster, in accordance with the environmental conditions. It is necessary to establish a ban season according to reproductive cycle. This study presents the gonadic cycle for an oyster population of Mecoacin lagoon along a year. It was defined five phases of the gonad development: resting, gametogenesis, mature or ripe, spawn and post spawn. Gametogenesis is present all the year, except December. The spawning activity was detected all year, except July and August. The ripe phase presented maximum values in August and December. It was proposed a modification of the ban seasons from April 15 - May 30, and September 15 - October 30 to March 15 - May 15 and September 1 - October 30, respectively, according to gonadic cycle obtained in this work. This modification would to avoid capture mature organisms (ready to spawn).     

Molecular characterization and expression analysis of an inhibitor of NF-κB (IκB) from Asiatic hard clam Meretrix meretrix

Inhibitor of NF-κB (IκB) is an important member of Rel/NF-κB signaling pathway, which is an important mediator of immune responses in innate immune system. In this study, the IκB cDNA of hard clam Meretrix meretrix (designated as Mm-IκB) was cloned and characterized. The full-length cDNA of Mm-IκB was of 2098 bp, containing a 5' untranslated region (UTR) of 123 bp, a 3' UTR of 810 bp with a poly (A) tail, and an open reading frame (ORF) of 1164 bp encoding a polypeptide of 387 amino acids. The high similarity of Mm-IκB with other IκBs from invertebrates indicated that Mm-IκB should be a member of IκB family. Similar to most IκBs, Mm-IκB possessed all conserved features critical for the fundamental structure and function of IκBs, such as five ankyrin repeats and a conserved degradation motif (DS(44)RYSS(48)). Two PEST domains and a phosphorylation site motif (S(367)EEE(370)) at the C-terminus of Mm-IκB were identified. By quantitative real-time RT-PCR analysis, mRNA level of Mm-IκB was found to be most abundantly expressed in the tissues of mantle, gill and hepatopancreas, weakly expressed in muscle, foot and haemocyte. The Mm-IκB gene expression was significantly up-regulated at 24 h in haemocyte and at 12 h in gill after Vibrio anguillarum challenge, respectively. The results suggested the involvement of Mm-IκB in response against bacterial infection and further highlighted its functional importance in the immune system of M. meretrix.     

Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Activation of the IκB kinase complex by HTLV-1 Tax requires cytosolic factors involved in Tax-induced polyubiquitination

Activation of NF-κB by human T cell leukaemia virus type 1 Tax is thought to be crucial in T-cell transformation and the onset of adult T cell leukaemia. Tax activates NF-κB through activation of the IκB kinase (IKK) complex, similar to cytokine-induced NF-κB activation, which involves active signalling complex formation using polyubiquitin chains as a platform. Although polyubiquitination of Tax was reported to be required for IKK activation, most studies have been performed using intact cells, in which secondary NF-κB activation can be induced by various cytokines that are secreted due to Tax-mediated primary NF-κB activation. Therefore, a cell-free assay system, in which IKK can be activated by adding highly purified recombinant Tax to cytosolic extract, was used to analyse Tax-induced IKK activation. In contrast to the cytosolic extract, the purified IKK complex was not activated by Tax, whereas, it was efficiently activated by MEKK1, that does not require polyubiquitination to activate IKK. Moreover, Tax-induced IKK activation was blocked when the cytosolic extract was mixed with either lysine-free, methylated or K63R ubiquitin. These results obtained through our cell-free assay suggest that K63-linked polyubiquitination is critical, but linear polyubiquitination is dispensable or insufficient for Tax-induced IKK activation.     

Aminoparthenolides as novel anti-leukemic agents: Discovery of the NF-κB inhibitor,DMAPT (LC-1)

A series of aminoparthenolide analogs (6–37) were synthesized and evaluated for their anti-leukemic activity. Eight compounds exhibited good anti-leukemic activity with LD₅₀’s in the low μM range (1.5–3.0 μM). Compounds 16, 24 and 30 were the most potent compounds in the series, causing greater than 90% cell death at 10 μM concentration against primary AML cells in culture, with LD₅₀ values of 1.7, 1.8 and 1.6 μM.