The absence of IL-6 does not affect Th2 cell development in vivo, but does lead to impaired proliferation, IL-2 receptor expression, and B cell responses

The role of IL-6 in Th2 cell differentiation and response development after the injection of eggs from Schistosoma mansoni was investigated using wild-type (WT) and IL-6-/- mice. IL-6 was induced in the lymph nodes (LN) of WT mice within 24 h of egg injection, and IL-4 production by WT LN cells and CD4 T cells isolated 24 h after egg injection and stimulated in vitro was observed. In the absence of IL-6, this early production of IL-4 by LN cells and purified CD4 T cells was not abolished; although the level of IL-4 produced by IL-6-/- LN cells was similar to WT, IL-4 production by purified IL-6-/- CD4 T cells was reduced compared with WT. Despite the difference in CD4 T cell production of IL-4, the development of egg-specific Th2 cells 7 days after egg injection was not affected by the absence of IL-6. Nevertheless, Ab production was impaired and the in vitro proliferative response of whole LN cell populations, CD4 and CD8 T cells, and B cells from IL-6-/- mice was poor compared with WT. The proliferative defect in the IL-6-/- cells correlated with decreased IL-2R expression, and addition of exogenous IL-6 enhanced IL-2R expression as well as proliferation of LN cells from IL-6-/- mice. These studies demonstrate that Th2 differentiation and response development in vivo is not dependent on IL-6, but that Th-dependent and independent B cell responses are. Our results also emphasize the importance of IL-6 for lymphoproliferation, possibly through induction of IL-2R expression.     

Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.     

SARS-CoV-2 suppresses IFNβ production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNβ production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNβ-promoter activity, whereas all six genes induced a collapse in IFNβ mRNA levels, corresponding with suppressed IFNβ protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.     

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ～40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.     

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment.     

Vagal blockade does not prevent adrenocorticotropin, cortisol, and cardiopulmonary responses to thromboxane A2.

Previously, we reported that thromboxane A2 (TxA2) mediates heart rate, adrenocorticotropin (ACTH), cortisol, and blood gas responses, although the specific site of action was not identified. In the present study, we interrupted vagal nervous transmission in chronically instrumented conscious sheep and infused the TxA2 mimetic U46619 or saline into the carotid artery or U46619 into the vena cava to determine whether TxA2 acts at vagal afferent nerves. Heart rate increased in all three groups during vagal blockade, and responses were not different between groups. Carotid artery and intravenous infusions of U46619 resulted in an increase in blood pressure, but responses were not different between groups. PaO2 decreased in response to vagal blockade in all groups, and responses were not different among groups. Arterial pH increased and PaCO2 decreased during vagal blockade in response to carotid artery U46619 infusions but not in response to vagal blockade alone or combined with carotid artery saline or intravenous U46619. ACTH, cortisol, and hematocrit increased significantly in response to carotid artery infusions of U46619 during vagal blockade but not in response to carotid artery saline or intravenous U46619 infusions. In summary, carotid artery infusions of TxA2 mimetic result in ACTH, cortisol, PaCO2, pHa, and hematocrit responses that are not prevented by vagal blockade. We conclude that these responses are mediated at a site perfused by the carotid vasculature and not at a site innervated by the vagal nerves, findings consistent with the hypothesis that TxA2 acts on the brain to mediate cardiopulmonary and pituitary-adrenal responses.     

The PPARdelta agonist, GW501516, promotes fatty acid oxidation but has no direct effect on glucose utilisation or insulin sensitivity in rat L6 skeletal muscle cells

Peroxisome proliferator-activated receptor-delta (PPARdelta) activation enhances skeletal muscle fatty acid oxidation and improves whole body glucose homeostasis and insulin sensitivity. Recently, GW501516, a selective PPARdelta agonist, was reported to increase glucose uptake in human skeletal myotubes by an AMPK-dependent mechanism that may contribute to the improved glucose tolerance. Here, we demonstrate that whilst GW501516 increases expression of PGC-1alpha and CPT-1 and stimulates fatty-acid oxidation in L6 myotubes, it fails to enhance insulin sensitivity, AMPK activity or glucose uptake and storage. Our findings exclude sarcolemmal glucose transport as a potential target for the therapeutic action of PPARdelta agonists in skeletal muscle.     

The centrosomal, putative tumor suppressor protein TACC2 is dispensable for normal development, and deficiency does not lead to cancer

TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.     

The luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, has no direct effect on spermatogenesis in the rat

Treatment of intact rats with luteinizing hormone-releasing hormone (LHRH) agonists has been shown to produce atrophy of a variable number of testicular seminiferous tubules. These findings raised the question of a possible direct versus indirect action of LHRH agonists on spermatogenesis. To answer this question, we treated hypophysectomized rats with the LHRH agonist [D-Trp6, des-Gly-NH2(10)]-LHRH ethylamide, dihydrotestosterone (DHT), or a combination of these two compounds for a period of 1 mo. Treatment of hypophysectomized animals with the LHRH agonist alone had no significant effect on the atrophy of seminiferous tubules found after hypophysectomy. DHT, however, maintained spermatogenesis at 80% of the level seen in intact animals. When DHT and the LHRH agonist were administered in combination, the stimulatory effects of DHT were observed with no significant interference caused by the LHRH agonist. This study shows that an LHRH agonist has no direct effect on the morphology of the seminiferous tubules in the absence of the pituitary gland and strongly suggests that the atrophy observed in the testis after LHRH agonist treatment in intact animals is mediated by the LHRH agonist-induced changes in luteinizing hormone secretion and/or direct action of the peptide on Leydig cells.     

RYK, a catalytically inactive receptor tyrosine kinase, associates with EphB2 and EphB3 but does not interact with AF-6

RYK is an atypical orphan receptor tyrosine kinase that lacks detectable kinase activity. Nevertheless, using a chimeric receptor approach, we previously found that RYK can signal via the mitogen-activated protein kinase pathway. Recently, it has been shown that murine Ryk can bind to and be phosphorylated by the ephrin receptors EphB2 and EphB3. In this study, we show that human RYK associates with EphB2 and EphB3 but is not phosphorylated by them. This association requires both the extracellular and cytoplasmic domains of RYK and is not dependent on activation of the Eph receptors. It was also previously shown that AF-6 (afadin), a PDZ domain-containing protein, associates with murine Ryk. We show here that AF-6 does not bind to human RYK in vitro or in vivo. This suggests that there are significant functional differences between human and murine RYK. Further studies are required to determine whether RYK modulates the signaling of EphB2 and EphB3.     

Enforced expression of Bcl-2 restores the number of NK cells,but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes,in IL-2/IL-15 receptor beta-chain-deficient mice

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.     

The weak interaction of LcrV and TLR2 does not contribute to the virulence of Yersinia pestis

Yersinia pestis and the enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica share the virulence-antigen LcrV. Previously, using reverse genetics we have proven that LcrV contributes to the virulence of Y. enterocolitica serotype O:8 by inducing IL-10 via Toll-like receptor 2 (TLR2). However, both the ability of Y. pestis LcrV to activate TLR2 and a possible role of TLR2-dependent IL-10 induction by LcrV in Y. pestis are not yet known. To eliminate interference from additional protein sequences, we produced LcrVs without affinity tags from Y. pestis and from Y. enterocolitica O:8 (LcrVO:8). LcrVO:8 was much more potent in TLR2-activity than Y. pestis LcrV. To analyse the role of TLR2 in plague, we infected both wild-type and TLR2-/- mice subcutaneously with Y. pestis GB. While TLR2-/- mice exhibited lower blood levels of IL-10 (day 2 post-infection) and of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and MCP-1 (day 4) than wild-type mice, there was no significant difference in survival. The low TLR2-activity of Y. pestis LcrV and associated cytokine expression might explain why - in contrast to Y. enterocolitica O:8 infection - TLR2-deficient mice are not more resistant than wild-type mice in a bubonic plague model.     

Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes

Injections of cytosolic preparations from mammalian sperm into oocytes have been shown to trigger calcium [Ca2+]i oscillations and initiate activation of development. Recently, a protein isolated from hamster sperm has been suggested to be involved in the generation of these oscillations and it was named "oscillin." The human homologue of hamster oscillin is glucosamine 6-phosphate isomerase (GPI, EC no. 5.3.1.10), an enzyme so far described to be involved in hexose phosphate metabolism. To assess the role of GPI on Ca2+ signaling, a human recombinant protein was generated in a prokaryotic system and injected into fura-2-dextran-loaded metaphase II (MII) mouse oocytes. Injection of recombinant GPI failed to induce Ca2+ responses in 12/12 injected MII oocytes despite the fact that the recombinant GPI was active as assessed by an enzymatic assay. Injection of buffer (0/6 oocytes) or fructose-6-phosphate, a product of GPI enzymatic reaction (0/5 oocytes), also failed to initiate Ca2+ responses. Conversely, injections of sperm cytosolic factor induced [Ca2+]i oscillations in all 17/17 oocytes. In addition, injection of recombinant GPI or GPI mRNA failed to induce parthenogenetic activation (0/30 oocytes). Immunofluorescence studies using an anti-GPI polyclonal antibody (GK) resulted in localization of GPI to the sperm's equatorial region. Incubation of the GK antibody with sperm extracts failed to block the [Ca2+]i responses induced by these extracts. Moreover, near complete depletion of GPI from sperm fractions by immunoprecipitation did not impair the ability of these fractions to induce [Ca2+]i oscillations. In summary, our results support the role of a sperm cytosolic component(s) in the generation of [Ca2+]i oscillations during mammalian fertilization, although a protein other than GPI/oscillin is likely to be the active calcium releasing factor.     

The alpha 2 chain of type 1 collagen does not map to mouse chromosome 16 but maps close to the Met proto-oncogene on mouse chromosome 6

The gene locus for the alpha 2 chain of type 1 collagen (Cola-2) was previously assigned to chromosome 16. Here we demonstrate, utilising both somatic cell hybrid analysis and genetic linkage analysis, in an interspecific Mus domesticus x Mus spretus cross that Cola-2 fails to cosegregate with mouse chromosome 16, but is linked to the Met proto-oncogene on chromosome 6.     

The application of ascorbate or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea nodules to water stress

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5 mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. −1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.     

Prostaglandins and congeners. IX. The synthesis of 15-deoxy-16-, 17-, or 20-hydroxyprostaglandins and 15-deoxy-15-hydroxymethylprostaglandin E2.

Prostaglandin congeners wherein the 15-hydroxy group is moved to the C16, C17, or C20 position or is replaced by a hydroxymethyl group were prepared via the 1, 4-addition of a lithium trialkyl-trans-alkenyl alanate to an appropriate cyclopentenone. Several of the 16-hydroxy derivatives showed significant activity as constrictors of the isolated gerbil colon and in bronchodilator and anti-secretory assays.