Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.     

Gamma-irradiated influenza virus uniquely induces IFN-I mediated lymphocyte activation independent of the TLR7/MyD88 pathway

Background

We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that γ-irradiated SFV does not induce IFN-I and the associated lymphocyte activation.

Principal Findings

Here we report that, in contrast to SFV, γ-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only γ-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways.

Conclusions

Our data suggest an important mechanism for the recognition of γ-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of γ-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.     

Effect of elevated [CO2] on stem wood properties of mature Norway spruce grown at different soil nutrient availability

The objective of the present study was to investigate the interactive effects of elevated [CO₂] and soil nutrient availability on secondary xylem structure and chemical composition of 41‐year‐old Norway spruce (Picea abies (L.) Karst.) trees. The nonfertilized and irrigated‐fertilized trees were, for 3 years, continuously exposed to elevated [CO₂] in whole‐tree chambers. Elevated [CO₂] decreased concentrations of soluble sugars, acid‐soluble lignin and nitrogen in stem wood, but the effects were not consistent between sampling height and/or fertilization. The effect of 2*ambient [CO₂] on wood structure depended on the exposure year and/or fertilization. Radial lumen diameter decreased and annual ring width increased in the second year of exposure (1999) in elevated [CO₂]. In the latter, the CO₂ effect was significant only in the nonfertilized trees. Stem wood chemistry and structure were significantly affected by fertilization. Fertilization increased the concentrations of nitrogen and gravimetric lignin, annual ring width, and radial lumen diameter. Fertilization decreased C/N ratio, mean ring density, earlywood density, latewood density, cell wall thickness, cell wall index, and latewood percentage. We conclude that elevated [CO₂] had only minor effects on wood properties while fertilization had more marked effects and thus may affect ecosystem processes and suitability of wood for different end‐use purposes.     

Effects of growth differences due to geographic location and N-fertilisation on wood chemistry of Norway spruce

We studied the effect of long-term nitrogen fertilisation on wood chemistry at two boreal sites in Finland: the northern site (Kemijärvi) and the southern site (Heinola). N-fertilisation was repeated in five-year intervals from the 1960s. Norway spruce (Picea abies L. Karst.) trees that had been planted in 1938 and 1954, in the northern and the southern site, respectively, were harvested in October 2002. Altogether 20 trees, in five different size classes, either unfertilised or fertilised, were felled. Wood sections at breast height, consisting of five consecutive annual rings, from six (Kemijärvi) or five (Heinola) points with different distances from the pith were examined. Differences in growth between the northern and southern sites were marked in favour of the southern site. In the northern site fertilisation had clearly increased the diameter growth, while in the southern site fertilisation had no effect. Nitrogen fertilisation resulted in slight changes in wood chemistry that included increased nitrogen concentrations in the northern site and extractives in the southern site. Stem wood had higher concentrations of extractives, starch, and uronic acids, and lower concentration of cellulose, in the northern than in the southern site. Changes in the stem wood chemistry along radial axis were marked. The changes in wood chemistry are discussed in relation to the physiological function and also how the changes can influence the suitability of wood for different end-use purposes.     

Comparison of Different Standard Methods to Evaluate the Total Concentrations of Heavy Metals in Waste Rock

The mining industry is a very rapidly increasing production sector in Finland. Many new mines have been opened recently and others are in the planning phase. Mining legislation has been recently revised and compilation of an ID-directive (Industrial Emission Directive, 2010) required to implement environmental law revision is in progress. The aim of this study was to compare different digestion methods recommended by the Finnish Environmental Administration for threshold and guideline value determinations in the Finnish PIMA Decree (214/2007) and Government Decree (591/2006) concerning recovery of certain wastes in earth construction. Altogether six methods were tested, of which five were microwave-assisted acid digestion methods: ISO 11466, EPA Method 3051A (aqua region), EPA Method 3051A (HNO₃), EPA Method 3051A (HNO₃/HCl), and SFS-EN 13656, while one was a fusion method with lithium tetraborate (ASTM C 1391 - 95). The results showed that concentrations of heavy metals (As, Cd, Co, Cu, Ni, Pb, Sb, V, and Zn) can be determined reproducibly with standard methods, but the chemical method used can have a significant impact on the results. This conclusion is very important, because the measured heavy metal values can affect the classification and processing of waste materials, and thereby can significantly impact the cost of treatment.     

Premature birth and circadian preference in young adulthood: evidence from two birth cohorts

A preference for eveningness (being a “night owl”) and preterm birth (<37 weeks of gestation) are associated with similar adversities, such as elevated blood pressure, impaired glucose regulation, poorer physical fitness, and lower mood. Yet, it remains unclear if and how preterm birth is associated with circadian preference. The aim of this study was to assess this association across the whole gestation range, using both objective and subjective measurements of circadian preference.

Circadian preference was measured among 594 young adults (mean age 24.3 years, SD 1.3) from two cohorts: the ESTER study and the Arvo Ylppö Longitudinal Study. We compared 83 participants born early preterm (<34 weeks) and 165 late preterm (34 to <37 weeks) with those born at term (≥37 weeks, n = 346). We also compared very low birth weight (VLBW, <1500 g) participants with term-born controls. We obtained objective sleep data with actigraphs that were worn for a mean period of 6.8 (SD 1.4) nights. Our primary outcome was sleep midpoint during weekdays and weekend. The sleep midpoint is the half-way time between falling asleep and waking up, and it represents sleep timing. We also investigated subjective chronotype with the Morningness–Eveningness Questionnaire (MEQ) in 688 (n = 138/221/329) ESTER participants. The MEQ consists of 19 questions, which estimates the respondent to be of a “morning”, “evening,” or “intermediate” chronotype, based on the Morningness–Eveningness Score (MES). We analyzed the data from the actigraphs and the MES with three linear regression models, and analyzed distribution of the chronotype class with Pearson χ2.

There were no consistent differences across the study groups in sleep midpoint. As compared with those born at term, the mean differences in minutes:seconds and 95% confidence intervals for the sleep midpoint were: early preterm weekdays 11:47 (?8:34 to 32:08), early preterm weekend 4:14 (?19:45 to 28:13), late preterm weekdays ?10:28 (?26:16 to 5:21), and late preterm weekend ?1:29 (?20:36 to 17:37). There was no difference in sleep timing between VLBW-participants and controls either. The distribution of chronotype in the MEQ among all participants was 12.4% morningness, 65.4% intermediate, and 22.2% eveningness. The distribution of the subjective chronotype class did not differ between the three gestational age groups (p = 0.98). The linear regression models did not show any influence of gestational age group or VLBW status on the MES (all p > 0.5).

We found no consistent differences between adults born early or late preterm and those born at term in circadian preference. The earlier circadian preference previously observed in those born smallest is unlikely to extend across the whole range of preterm birth.     

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

Background

Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.

Methods and Findings

Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB⁺ Tc cells) or granzyme A and granzyme B deficient (GzmA×B^−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.

Significance

Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

The 11q;22q translocation: A European collaborative analysis of 43 cases

Summary Translocation between the long arms of chromosomes 11 and 22 is usually detected in offspring with an unbalanced karyotype following a 3:1 disjunction resulting in partial trisomy. Since by the end of 1976 it was suspected that this translocation might be more frequent than one would deduce from published reports, it was decided to call for a collaborative effort in Europe to collect unpublished cases. In response, 42 cases were collected in Europe, and one case from New Zealand was added. The following countries were represented with the number of cases indicated in parentheses: Czechoslovakia (2), Denmark (4), Finland (3), France (6), Germany (1), Italy (5), The Netherlands (9), Sweden (6), United Kingdom (4), Yugoslavia (2). The wide geographical distribution indicates a multifocal origin of the translocation. Among the unpublished cases, 31 were ascertained as unbalanced carriers [47,XX or XY,+der(22),t(11;22)] and 12 as balanced balanced carriers [46,XX and XY,t(11;22)]. Among the published cases, 10 were ascertained in unbalanced and 3 in balanced carriers. The breakpoints of the translocations indicated by the contributors varied, the most frequently reported being 11q23;22q11 (25 cases), followed by q25;q13 (10 cases). While the first one seems more likely, it was not possible to decide whether the breakpoints were the same in all cases.All 32 probands with unbalanced karyotypes had inherited the translocation, 31 from the mother and only 1 from the father. This ratio became 43:1 when the published cases were added. A segregation analysis revealed that in families ascertained through probands with unbalanced karyotypes there was a ratio of carriers to normal (all karyotyped) 54:55, not a significant difference. The formal maximum (minimum) recurrence risk for this unbalanced translocation was calculated to be 5.6% (2.7%). When the ascertainment was through a balanced proband, the maximum risk was 2.7%. The risk was calculated as 5.7% for female and 4.3% for male carriers. The mean family size was 1.67 for the offspring of female carriers and 0.78 for the offspring of male carriers. This significant difference suggests that heterozygosity for the translocation reduces fertility in males. Indeed, several of the probands with balanced karyotypes were ascertained because of sub- or infertility. Only 2 de novo translocations were found among the 59 probands, and both, were among the 12 cases ascertained as balanced carriers. The source, quality, and quantity of the clinical data for the subjects with unbalanced karyotypes were variable, and no definite conclusions were possible about phenotypes. The following signs were recorded in 10 or more of the 45 cases: low birth weight, delayed psychomotor development, hypotonia, microcephaly, craniofacial asymmetry, malformed ears with pits and tags, cleft palate, micro-/retrognathia, large beaked nose, strabismus, congenital heart disease, cryptorchidism, and congenital dislocation of the hip joints. Many signs were similar to those considered typical of trisomy 11q, and the phenotype coincided almost completely with the presumptive phenotype of complete trisomy 22. No cases with coloboma was recorded, while other signs of the cat-eye syndrome were found in several probands. This might indicate that individuals with the cat-eye syndrome and carriers of the unbalanced 11/22 translocation have the same segment of 22 in triplicate plus or minus another chromosome segment.     

Influence of the menstrual cycle and oral contraceptives on thermoregulatory responses to exercise in young women

Thermoregulatory responses to exercise in relation to the phase of the menstrual cycle were studied in ten women taking oral contraceptives (P) and in ten women not taking oral contraceptives (NP). Each subject was tested for maximal aerobic capacity ( ) and for 50% exercise in the follicular (F) and luteal (L) phases of the menstrual cycle. Since the oral contraceptives would have prevented ovulation a quasi-follicular phase (q-F) and a quasi-luteal phase (q-L) of the menstrual cycle were assumed for P subjects. Exercise was performed on a cycle ergometer at an ambient temperature of 24° C and relative air humidity of 50%. Rectal (T _re), mean skin ( ), mean body ( ) temperatures and heart rate (f _c) were measured. Sweat rate was estimated by the continuous measurement of relative humidity of air in a ventilated capsule placed on the chest, converted to absolute pressure (PH₂O_chest). Gain for sweating was calculated as a ratio of increase inPH₂O_chest to the appropriate increase inT _re for the whole period of sweating (G) and for unsteady-state (G_u) separately. The did not differ either between the groups of subjects or between the phases of the menstrual cycle. In P, rectal temperature threshold for sweating (T _{re, td}) was 37.85° C in q-L and 37.60° C in q-F (P < 0.01) and corresponded to a significant difference fromT _re at rest. TheT _re, andf _c increased similarly during exercise in q-F and q-L. No menstrual phase-related differences were observed either in the dynamics of sweating or in G. In NP,T _{re, td} was shorter in L than in F (37.70 vs 37.47° C,P<0.02) with a significantly greater value fromT _re at rest. The dynamics and G for sweating were also greater in L than in F. The G_u was 36.8 versus 16.6 kPa · ° C^–1 (P<0.01) while G was 6.4 versus 3.8 kPa · ° C^–1 (P<0.05), respectively. TheT _re, andf _c increased significantly more in phase F than in phase L. It was concluded that in these women performing moderate exercise, there was a greater temperature threshold and larger gains for sweating in phase L than in phase F. Intake of oral contraceptives reduced the differences in the gains for sweating making the thermoregulatory responses to exercise more uniform.