Programmed cell death during development of cowpea (Vigna unguiculata (L.) Walp.) seed coat

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase‐like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events.     

Induction of autoantibodies against lung matrix proteins and smoke-induced inflammation in mice

Background

Despite recommendations for outpatient management, low risk patients with lower respiratory tract infections (LRTIs) are often hospitalized. This survey analyzed perceptions of physicians, nurses, patients and relatives about feasibility of outpatient management and required duration of hospital stay.

Methods

We performed a prospective, observational questionnaire survey in hospitalized patients with LRTI as part of a multicenter trial. Treating physicians and nurses, patients and their relatives were asked on admission and before discharge about feasibility of outpatient treatment over 5 dimensions (medical, nursing, organizational factors, and patients' and relatives' preferences) using continuous scales.

Results

On admission, 12.6% of physicians, 15.1% of nurses, 18.0% of patients and 5.2% of relatives believed that outpatient treatment would be possible. Before hospital discharge, 31.1% of physicians, 32.2% of nurses, 11.6% of patients and 4.1% of relatives thought that earlier discharge would have been feasible. Medical factors were the most frequently perceived motives for inpatient management. These perceptions were similar in all LRTI subgroups and independent of disease severity and associated expected mortality risks as assessed by the Pneumonia Severity Index (PSI).

Conclusion

Independent of type and severity of respiratory tract infection, the misperceived high severity and expected mortality and morbidity were the predominant reasons why treating physicians, nurses, patients and their relatives unanimously believed that inpatient management was necessary. Better assessment and communication about true expected medical risks might contribute to a pathway to shorten in-hospital days and to introduce a more risk-targeted and individually tailored allocation of health-care resources.

Trial Registration

NCT00350987     

Mapping the Naked Neck (NA) and Polydactyly (PO) mutants of the chicken with microsatellite molecular markers

The bulked segregant analysis methodology has been used to map, with microsatellite markers, two morphological mutations in the chicken: polydactyly (PO) and naked neck (NA). These autosomal mutations show partial dominance for NA, and dominance with incomplete penetrance for PO. They were mapped previously to different linkage groups of the classical map, PO to the linkage group IV and NA being linked to the erythrocyte antigen CPPP. An informative family of 70 offspring was produced by mating a sire, heterozygous for each of the mutations, to 7 dams homozygous recessive for each locus. Three DNA pools were prepared, pool PO included 20 chicks exhibiting at least one extra-toe, pool NA included 20 non-polydactyly chicks showing the typical phenotype associated with heterozygosity for the naked neck mutation, and pool NP included 20 chicks exhibiting neither of the mutant phenotypes. Typings were done on an ABI-373 automatic sequencer with 147 microsatellite markers covering most of the genome. An unbalanced distribution of sire marker alleles were detected between pool PO, and pools NA and NP, for two markers of chromosome 2p, MCW0082 and MCW0247. A linkage analysis taking into account the incomplete penetrance of polydactyly (80%) was performed with additional markers of this region and showed that the closest marker to the PO locus was MCW0071 (5 cM, lod score = 9). MCW0071 lies within the engrailed gene EN2 in the chicken. In the mouse, the homologous gene maps on chromosome 5, close to the hemimelic extra-toes mutation Hx. In the case of the NA locus, markers of chromosome 3 were selected because CPPP was mapped on this chromosome. Analysis of individual typings showed a linkage of 5.7 cM (lod score = 13) between the NA locus and ADL0237 in the distal region of chromosome 3q. These results contribute to connecting the former classical map to the molecular genetic map of the chicken, and open the way to the identification of the molecular nature of two developmental mutations of the chicken that are known to occur in many breeds of chickens.     

Effect of fertilizer levels on grain yield,incidence and severity of downy mildew in pearl millet

The effect of various levels of nitrogen (0.0, 30.0, 60.0, 120.0) and phosphorus (0.0, 6.5, 13.0, 36.0) on the incidence and severity of downy mildew of pearl millet and yield of two pearl millet varieties (Zango and GB8375) were studied under field conditions in 2000 and 2001 respectively. Both nitrogen and phosphorus significantly increased incidence and severity of the disease in the two varieties. Grain yield and 1000 grain weight of the varieties also increased with nitrogen and phosphorus levels.     

On the relationship between an Asian haplotype on chromosome 6 that reduces androstenone levels in boars and the differential expression of SULT2A1 in the testis

Background

Androstenone is one of the major compounds responsible for boar taint, a pronounced urine-like odor produced when cooking boar meat. Several studies have identified quantitative trait loci (QTL) for androstenone level on Sus scrofa chromosome (SSC) 6. For one of the candidate genes in the region SULT2A1, a difference in expression levels in the testis has been shown at the protein and RNA level.

Results

Haplotypes were predicted for the QTL region and their effects were estimated showing that haplotype 1 was consistently related with a lower level, and haplotype 2 with a higher level of androstenone. A recombinant haplotype allowed us to narrow down the QTL region from 3.75 Mbp to 1.94 Mbp. An RNA-seq analysis of the liver and testis revealed six genes that were differentially expressed between homozygotes of haplotypes 1 and 2. Genomic sequences of these differentially expressed genes were checked for variations within potential regulatory regions. We identified one variant located within a CpG island that could affect expression of SULT2A1 gene. An allele-specific expression analysis in the testis did not show differential expression between the alleles of SULT2A1 located on the different haplotypes in heterozygous animals. However a synonymous mutation C166T (SSC6: 49,117,861 bp in Sscrofa 10.2; C/T) was identified within the exon 2 of SULT2A1 for which the haplotype 2 only had the C allele which was higher expressed than the T allele, indicating haplotype-independent allelic-imbalanced expression between the two alleles. A phylogenetic analysis for the 1.94 Mbp region revealed that haplotype 1, associated with low androstenone level, originated from Asia.

Conclusions

Differential expression could be observed for six genes by RNA-seq analysis. No difference in the ratio of C:T expression of SULT2A1 for the haplotypes was found by the allele-specific expression analysis, however, a difference in expression between the C over T allele was found for a variation within SULT2A1, showing that the difference in androstenone levels between the haplotypes is not caused by the SNP in exon 2.     

Analysis of bronchoalveolar lavage fluid proteome from systemic sclerosis patients with or without functional, clinical and radiological signs of lung fibrosis

Lung fibrosis is a major cause of mortality and morbidity in systemic sclerosis (SSc). However, its pathogenesis still needs to be elucidated. We examined whether the alteration of certain proteins in bronchoalveolar lavage fluid (BALF) might have a protective or a causative role in the lung fibrogenesis process. For this purpose we compared the BALF protein profile obtained from nine SSc patients with lung fibrosis (SSc_Fib+) with that obtained from six SSc patients without pulmonary fibrosis (SSc_Fib-) by two-dimensional gel electrophoresis (2-DE). Only spots and spot-trains that were consistently expressed in a different way in the two study groups were taken into consideration. In total, 47 spots and spot-trains, corresponding to 30 previously identified proteins in human BALF, showed no significant variation between SSc_Fib+ patients and SSc_Fib- patients, whereas 24 spots showed a reproducible significant variation in the two study groups. These latter spots corresponded to 11 proteins or protein fragments, including serum albumin fragments (13 spots), 5 previously recognized proteins (7 spots), and 4 proteins (3 spots) that had not been previously described in human BALF maps, namely calumenin, cytohesin-2, cystatin SN, and mitochondrial DNA topoisomerase 1 (mtDNA TOP1). Mass analysis did not determine one protein-spot. The two study groups revealed a significant difference in BALF protein composition. Whereas levels of glutathione S-transferase P (GSTP), Cu–Zn superoxide dismutase (SOD) and cystatin SN were downregulated in SSc_Fib+ patients compared with SSc_Fib- patients, we observed a significant upregulation of α1-acid glycoprotein, haptoglobin-α chain, calgranulin (Cal) B, cytohesin-2, calumenin, and mtDNA TOP1 in SSc_Fib+ patients. Some of these proteins (GSTP, Cu–Zn SOD, and cystatin SN) seem to be involved in mechanisms that protect lungs against injury or inflammation, whereas others (Cal B, cytohesin-2, and calumenin) seem to be involved in mechanisms that drive lung fibrogenesis. Even if the 2-DE analysis of BALF did not provide an exhaustive identification of all BALF proteins, especially those of low molecular mass, it allows the identification of proteins that might have a role in lung fibrogenesis. Further longitudinal studies on larger cohorts of patients will be necessary to assess their usefulness as predictive markers of disease.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Increased levels of (class switched) memory B cells in peripheral blood of current smokers

There is increasing evidence that a specific immune response contributes to the pathogenesis of COPD. B-cell follicles are present in lung tissue and increased anti-elastin titers have been found in plasma of COPD patients. Additionally, regulatory T cells (Tregs) have been implicated in its pathogenesis as they control immunological reactions. We hypothesize that the specific immune response in COPD is smoke induced, either by a direct effect of smoking or as a result of smoke-induced lung tissue destruction (i.e. formation of neo-epitopes or auto antigens). Furthermore, we propose that Tregs are involved in the suppression of this smoke-induced specific immune response.The presence of B cells, memory B cells and Tregs was assessed by flow cytometry in peripheral blood of 20 COPD patients and 29 healthy individuals and related to their current smoking status.COPD patients had lower (memory) B-cell percentages and higher Treg percentages in peripheral blood than healthy individuals, with a significant negative correlation between these cells. Interestingly, current smokers had higher percentages of (class-switched) memory B cells than ex-smokers and never smokers, irrespective of COPD.This increase in (class-switched) memory B cells in current smokers is intriguing and suggests that smoke-induced neo-antigens may be constantly induced in the lung. The negative correlation between B cells and Tregs in blood is in line with previously published observations that Tregs can suppress B cells. Future studies focusing on the presence of these (class switched) memory B cells in the lung, their antigen specificity and their interaction with Tregs are necessary to further elucidate the specific B-cell response in COPD.