Genetic polymorphisms associated with rubella virus-specific cellular immunity following MMR vaccination

Rubella virus causes a relatively benign disease in most cases, although infection during pregnancy can result in serious birth defects. An effective vaccine has been available since the early 1970s and outbreaks typically do not occur among highly vaccinated (≥2 doses) populations. Nevertheless, considerable inter-individual variation in immune response to rubella immunization does exist, with single-dose seroconversion rates ~95 %. Understanding the mechanisms behind this variability may provide important insights into rubella immunity. In the current study, we examined associations between single nucleotide polymorphisms (SNPs) in selected cytokine, cytokine receptor, and innate/antiviral genes and immune responses following rubella vaccination in order to understand genetic influences on vaccine response. Our approach consisted of a discovery cohort of 887 subjects aged 11–22 at the time of enrollment and a replication cohort of 542 older adolescents and young adults (age 18–40). Our data indicate that SNPs near the butyrophilin genes (BTN3A3/BTN2A1) and cytokine receptors (IL10RB/IFNAR1) are associated with variations in IFNγ secretion and that multiple SNPs in the PVR gene, as well as SNPs located in the ADAR gene, exhibit significant associations with rubella virus-specific IL-6 secretion. This information may be useful, not only in furthering our understanding immune responses to rubella vaccine, but also in identifying key pathways for targeted adjuvant use to boost immunity in those with weak or absent immunity following vaccination.     

Synaptic underpinnings of altered hippocampal function in glutaminase-deficient mice during maturation

Glutaminase-deficient mice (GLS1 hets), with reduced glutamate recycling, have a focal reduction in hippocampal activity, mainly in CA1, and manifest behavioral and neurochemical phenotypes suggestive of schizophrenia resilience. To address the basis for the hippocampal hypoactivity, we examined synaptic plastic mechanisms and glutamate receptor expression. Although baseline synaptic strength was unaffected in Schaffer collateral inputs to CA1, we found that long-term potentiation was attenuated. In wild-type (WT) mice, GLS1 gene expression was highest in the hippocampus and cortex, where it was reduced by about 50% in GLS1 hets. In other brain regions with lower WT GLS1 gene expression, there were no genotypic reductions. In adult GLS1 hets, NMDA receptor NR1 subunit gene expression was reduced, but not AMPA receptor GluR1 subunit gene expression. In contrast, juvenile GLS1 hets showed no reductions in NR1 gene expression. In concert with this, adult GLS1 hets showed a deficit in hippocampal-dependent contextual fear conditioning, whereas juvenile GLS1 hets did not. These alterations in glutamatergic synaptic function may partly explain the hippocampal hypoactivity seen in the GLS1 hets. The maturity-onset reduction in NR1 gene expression and in contextual learning supports the premise that glutaminase inhibition in adulthood should prove therapeutic in schizophrenia.     

A New C-Terminal Cleavage Product of Procaspase-8, p30, Defines an Alternative Pathway of Procaspase-8 Activation

Caspase-8 is the main initiator caspase in death receptor-induced apoptosis. Procaspase-8 is activated at the death-inducing signaling complex (DISC). Previous studies suggested a two-step model of procaspase-8 activation. The first cleavage step occurs between the protease domains p18 and p10. The second cleavage step takes place between the prodomain and the large protease subunit (p18). Subsequently, the active caspase-8 heterotetramer p18₂-p10₂ is released into the cytosol, starting the apoptotic signaling cascade. In this report, we have further analyzed procaspase-8 processing upon death receptor stimulation directly at the DISC and in the cytosol. We have found an alternative sequence of cleavage events for procaspase-8. We have demonstrated that the first cleavage can also occur between the prodomain and the large protease subunit (p18). The resulting cleavage product, p30, contains both the large protease subunit (p18) and the small protease subunit (p10). p30 is further processed to p10 and p18 by active caspases. Furthermore, we show that p30 can sensitize cells toward death receptor-induced apoptosis. Taken together, our data suggest an alternative mechanism of procaspase-8 activation at the DISC.Apoptosis can be triggered by a number of factors, including UV or γ-irradiation, chemotherapeutic drugs, and signaling from death receptors (11, 12). CD95 (APO-1/Fas) is a member of the death receptor family, a subfamily of the tumor necrosis factor receptor (TNF-R) superfamily (1, 30). Eight members of the death receptor subfamily have been characterized so far: TNF-R1 (DR1, CD120a, p55, p60), CD95 (DR2, APO-1, Fas), DR3 (APO-3, LARD, TRAMP, WSL1), TRAIL-R1 (APO-2, DR4), TRAIL-R2 (DR5, KILLER, TRICK2), DR6, EDA-R, and NGF-R (13). Cross-linking of CD95 by its natural ligand, CD95L (CD178) (29), or by agonistic antibodies induces apoptosis in sensitive cells (31, 36). The death-inducing signaling complex (DISC) is formed within seconds after CD95 stimulation (9). The DISC consists of oligomerized, probably trimerized CD95 receptors, the adaptor molecule FADD, two isoforms of procaspase-8 (procaspase-8a and -8b), procaspase-10, and c-FLIP_L/S/R (6, 19, 21, 25, 27). The interactions between molecules at the DISC are based on homotypic contacts. The death domain of the receptor interacts with the death domain of FADD, while the death effector domain (DED) of FADD interacts with the N-terminal tandem DEDs of procaspase-8 and -10 and c-FLIP_L/S/R.Two isoforms of procaspase-8 (procaspase-8a and procaspase-8b) were reported to be bound to the DISC (24). Both isoforms possess two tandem DEDs, as well as the catalytic subunits p18 and p10 (see Fig. ​Fig.1A).1A). Procaspase-8a contains an additional 2-kDa (15-amino-acid [aa]) fragment, which results from the translation of exon 9. This small fragment is located between the second DED and the large catalytic subunit, resulting in different lengths of procaspase-8a and -8b (p55 and p53 kDa), respectively.Open in a separate windowFIG. 1.A new 30-kDa protein is detected by the anti-caspase-8 MAb C15. (A) Scheme of procaspase-8 and its cleavage products. The binding sites of the anti-caspase-8 MAbs C5 and C15 are indicated. (B) The B-lymphoblastoid cell lines SKW6.4, Raji, and BJAB and the T-cell lines CEM, Jurkat 16, and caspase-8-deficient Jurkat (clone JI9.2) were stimulated with LZ-CD95L for the indicated times, followed by caspase-8 immunoprecipitation (C8-IP) using the anti-caspase-8 MAb C15 directed against the p18 subunit of procaspase-8. Western blotting of immunoprecipitates was performed using the anti-caspase-8 MAb C15 (**, Ig heavy chain; *, unspecific band). (C) SKW6.4 cells were stimulated with LZ-CD95L for different times, and procaspase-8 processing in total cellular lysates was analyzed by Western blotting using the anti-caspase-8 MAb C15. (D) B-lymphoblastoid BJAB cells were stimulated with LZ-TRAIL for different times, and procaspase-8 processing was analyzed as described for panel C. (E) Primary human T cells (day 6) were stimulated with LZ-CD95L, and procaspase-8 processing was analyzed as described for panel C (*, unspecific band).Activation of procaspase-8 is believed to follow an “induced-proximity” model in which high local concentrations and a favorable mutual orientation of procaspase-8 molecules at the DISC lead to their autoproteolytic processing (2, 3, 20). There is strong evidence from several in vitro studies that autoproteolytic activation of procaspase-8 occurs after oligomerization at the receptor complex (20). Furthermore, it has been shown that homodimers of procaspase-8 have proteolytic activity and that proteolytic processing of procaspase-8 occurs between precursor homodimers (3).Procaspase-8a/b (p55/p53) processing at the DISC has been described to involve two sequential cleavage steps (see Fig. ​Fig.1A).1A). This process is referred to as the “two-step model” (3, 17). The first cleavage step occurs between the two protease domains, and the second cleavage step takes place between the prodomain and the large protease subunit (see Fig. ​Fig.1A)1A) (15). During the first cleavage step, the cleavage at Asp³⁷⁴ generates the two subunits p43/p41 and p12. Both cleavage products remain bound to the DISC: p43/p41 by DED interactions and p12 by interactions with the large protease domain of p43/p41. The second cleavage step takes place at Asp²¹⁶ and Asp³⁸⁴, producing the active enzyme subunits p18, p10, and the prodomain p26/p24. As a result of procaspase-8 processing, the active caspase-8 heterotetramer p18₂-p10₂ is formed at the DISC. This heterotetramer is subsequently released into the cytosol, starting the apoptotic signaling cascade (14).Recent studies have shown that processing of procaspase-8 at the DISC is more complicated and can involve additional steps like the generation of a prolonged prodomain of procaspase-8, termed CAP3 (p27), that is quickly converted to p26 (see Fig. ​Fig.1A)1A) (7).In addition to its central role in death receptor-induced apoptosis, caspase-8 was reported to be required for proliferation of lymphocytes (12, 23). Recently caspase-8 was shown to be an important factor for NF-κB activation following T-cell receptor stimulation (28). The mechanism underlying the dual role of caspase-8 activity and its regulation is largely unknown.In the present study, we show that upon death receptor stimulation, p30 is formed by cleavage at Asp²¹⁰, a yet-unknown cleavage product of procaspase-8, which comprises the C terminus of procaspase-8. p30 turned out to be a key intermediate product in the course of procaspase-8 processing. Furthermore, we suggest that the p30-mediated activation of procaspase-8 plays an important role in the amplification of the death signal. Taken together, our findings provide a new mechanism of procaspase-8 activation and extend the current two-step cleavage model by an alternative activation pathway.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Seed morphology of Rhododendron sichotense (Ericaceae): systematic implications

The size of Rhododendron sichotense seeds and exotesta cells from 20 populations were studied using a scanning electron microscope (SEM). The results of a correlation analysis showed that seed size depends on bioclimatic factors. As a species marker, we used the exotesta cell elongation coefficient. This characteristic was unresponsive to environmental variables, demonstrating its taxonomic significance. In the centre of the species range, the samples had an exotesta cell elongation coefficient that was typical for the species, but in samples from the far northern and southern parts of the range, where the distribution of R. sichotense meets that of of R. dauricum L. and R. mucronulatum Turcz., respectively, the value of the exotesta cell elongation coefficient was intermediate between these species. Therefore, the exotesta cell elongation coefficient can be recommended for use in the detection of hybridisation areas.     

Trapping crystal nucleation of cholesterol monohydrate: relevance to pathological crystallization

Crystalline nucleation of cholesterol at the air-water interface has been studied via grazing incidence x-ray diffraction using synchrotron radiation. The various stages of cholesterol molecular assembly from monolayer to three bilayers incorporating interleaving hydrogen-bonded water layers in a monoclinic cholesterol.H(2)O phase, has been monitored and their structures characterized to near atomic resolution. Crystallographic evidence is presented that this multilayer phase is similar to that of a reported metastable cholesterol phase of undetermined structure obtained from bile before transformation to the triclinic phase of cholesterol.H(2)O, the thermodynamically stable macroscopic form. According to grazing incidence x-ray diffraction measurements and crystallographic data, a transformation from the monoclinic film structure to a multilayer of the stable monohydrate phase involves, at least initially, an intralayer cholesterol rearrangement in a single-crystal-to-single-crystal transition. The preferred nucleation of the monoclinic phase of cholesterol.H(2)O followed by transformation to the stable monohydrate phase may be associated with an energetically more stable cholesterol bilayer arrangement of the former and a more favorable hydrogen-bonding arrangement of the latter. The relevance of this nucleation process of cholesterol monohydrate to pathological crystallization of cholesterol from cell biomembranes is discussed.     

On the mechanisms of the reaction of dodecatungstophosphate with alkyl radicals in aqueous solutions

The reduced lacunary polyoxotungstate, [PW₁₁O₃₉]⁸⁻, reacts with the ^.CH₂CH(OH)CH₃ and ^.CH₂C(CH₃)₂OH radicals via a mechanism involving β-hydroxide elimination to yield propene and 2-methyl propene respectively, and [PW₁₁O₃₉]⁷⁻. [PW₁₁O₃₉]⁸⁻ is also oxidized by methyl radicals in a reaction which yields methane as the major product. It is proposed that the reactions proceed via the formation of short lived transients with W-C σ bonds.