Regulation of etioplast pigment-protein complexes, inner membrane architecture, and protochlorophyllide a chemical heterogeneity by light-dependent NADPH:protochlorophyllide oxidoreductases A and B

The etioplast of dark-grown angiosperms is characterized by the prolamellar body (PLB) inner membrane, the absence of chlorophyll, and the accumulation of divinyl and monovinyl derivatives of protochlorophyll(ide) a [Pchl(ide) a]. Either of two structurally related, but differentially expressed light-dependent NADPH:Pchlide oxidoreductases (PORs), PORA and PORB, can assemble the PLB and form dark-stable ternary complexes containing enzymatically photoactive Pchlide-F655. Here we have examined in detail whether these polypeptides play redundant roles in etioplast differentiation by manipulating the total POR content and the PORA-to-PORB ratio of etiolated Arabidopsis seedlings using antisense and overexpression approaches. POR content correlates closely with PLB formation, the amounts, spectroscopic properties, and photoreduction kinetics of photoactive Pchlide, the ratio of photoactive Pchlide-F655 to non-photoactive Pchl(ide)-F632, and the ratio of divinyl- to monovinyl-Pchl(ide). This last result defines POR as the first endogenous protein factor demonstrated to influence the chemical heterogeneity of Pchl(ide) in angiosperms. It is intriguing that excitation energy transfer between different spectroscopic forms of Pchl(ide) in etiolated cotyledons remains largely independent of POR content. We therefore propose that the PLB contains a minimal structural unit with defined pigment stoichiometries, within which a small amount of non-photoactive Pchl(ide) transfers excitation energy to a large excess of photoactive Pchlide-F655. In addition, our data suggests that POR may bind not only stoichiometric amounts of photoactive Pchlide, but also substoichiometric amounts of non-photoactive Pchl(ide). We conclude that the typical characteristics of etioplasts are closely related to total POR content, but not obviously to the specific presence of PORA or PORB.     

THE ORIGIN OF WEST EUROPEAN SUBSPECIES OF HONEYBEES (APIS MELLIFERA): NEW INSIGHTS FROM MICROSATELLITE AND MITOCHONDRIAL DATA

Apis mellifera is composed of three evolutionary branches including mainly African (branch A), western and northern European (branch M), and southeastern European (branch C) populations. The existence of morphological clines extending from the equator to the Polar Circle through Morocco and Spain raised the hypothesis that the branch M originated in Africa. Mitochondrial DNA analysis revealed that branches A and M were characterized by highly diverged lineages implying very remote links between both branches. It also revealed that mtDNA haplotypes from lineages A coexisted with haplotypes M in the Iberian Peninsula and formed a south-north frequency cline, suggesting that this area could be a secondary contact zone between the two branches. By analyzing 11 populations sampled along a France-Spain/Portugal-Morocco-Guinea transect at 8 microsatellite loci and the DraI RFLP of the COI-COII mtDNA marker, we show that Iberian populations do not present any trace of “africanization” and are very similar to French populations when considering microsatellite markers. Therefore, the Iberian Peninsula is not a transition area. The higher haplotype A variability observed in Spanish and Portuguese samples compared to that found in Africa is explained by a higher mutation rate and multiple and recent introductions. Selection appears to be the best explanation to the morphological and allozymic clines and to the diffusion and maintenance of African haplotypes in Spain and Portugal.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Tonoplast intrinsic proteins from cauliflower (Brassica oleracea L. var. botrytis): immunological analysis, cDNA cloning and evidence for expression in meristematic tissues

The vacuolar membrane (tonoplast) of plant cells contains aquaporins, protein channels that facilitate the selective transport of water. These tonoplast intrinsic proteins (TIPs) of 23–29 kDa belong to the ancient major intrinsic protein (MIP) family. A monospecific polyclonal antiserum directed against a 26 kDa intrinsic protein from the tonoplast of meristematic cells from cauliflower (Brassica oleracea L. var. botrytis) was used to screen a cDNA library. Two distinct cDNAs have been isolated. Both clones, c26-1 and c26-2, encode closely related TIPs. The c26-1 insert, consisting of 933 bp upstream of the poly(A) tail, is a full-length cDNA with an open reading frame encoding a protein of 251 amino acids with a calculated M_r of 25 500. The c26-2 insert is a 5′ truncated cDNA. The two cDNAs share 90.5% sequence identity within their overlapping coding regions but only 35% sequence identity in the 3′␣untranslated regions, indicating that highly related TIP-encoding genes are expressed in meristematic cells. Although TIPs have previously been found in a variety of cell types, they have not been found in meristems. The derived amino acid sequences (BobTIP26-1 and BobTIP26-2, respectively) closely resemble the aquaporin γ-TIP from Arabidopsis thaliana. Northern blot analysis and in situ hybridization show that BobTIP26 mRNAs preferentially accumulate in highly meristematic cells, mostly before and during cell enlargement, and in the living cells of the xylem. This differential pattern of expression is also found by immunodetection of BobTIP26 polypeptides. The gene expression patterns are discussed with respect to the probable function of the gene products. Received: 27 March 1997 / Accepted: 20 May 1997     

N-Terminal 2,3-diaminopropionic acid (Dap) peptides as efficient methylglyoxal scavengers to inhibit advanced glycation endproduct (AGE) formation

2,3-Diaminopropionic acid (Dap) and N-terminal Dap peptides have been found to inhibit in vitro protein-modifications by methylglyoxal (MG), one of the highly reactive α-dicarbonyl compounds. MG scavenging potency of the newly synthesized N-terminal Dap peptides is demonstrated by RP-HPLC, SDS–PAGE and non-denaturing PAGE analysis, assays for enzymatic activity and cell viability study and was compared with that of known AGE inhibitors, such as aminoguanidine, pyridoxamine, metformin and carnosine. Two addition products of MG and l-Dap-l-Leu are separated by HPLC and their chemical structures are characterized by ¹H and ¹³C NMR spectroscopy to indicate that both of them are pyrazines derived from 2 molecules of MG and 1 molecule of l-Dap-l-Leu. Mutagenic activities of l-Dap-l-Leu and l-Dap-l-Val and their metabolites according to the Ames assay are found to be negative.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

IL22/IL-22R Pathway Induces Cell Survival in Human Glioblastoma Cells

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.     

Nest Predation Deviates from Nest Predator Abundance in an Ecologically Trapped Bird

In human-modified environments, ecological traps may result from a preference for low-quality habitat where survival or reproductive success is lower than in high-quality habitat. It has often been shown that low reproductive success for birds in preferred habitat types was due to higher nest predator abundance. However, between-habitat differences in nest predation may only weakly correlate with differences in nest predator abundance. An ecological trap is at work in a farmland bird (Lanius collurio) that recently expanded its breeding habitat into open areas in plantation forests. This passerine bird shows a strong preference for forest habitat, but it has a higher nest success in farmland. We tested whether higher abundance of nest predators in the preferred habitat or, alternatively, a decoupling of nest predator abundance and nest predation explained this observed pattern of maladaptive habitat selection. More than 90% of brood failures were attributed to nest predation. Nest predator abundance was more than 50% higher in farmland, but nest predation was 17% higher in forest. Differences between nest predation on actual shrike nests and on artificial nests suggested that parent shrikes may facilitate nest disclosure for predators in forest more than they do in farmland. The level of caution by parent shrikes when visiting their nest during a simulated nest predator intrusion was the same in the two habitats, but nest concealment was considerably lower in forest, which contributes to explaining the higher nest predation in this habitat. We conclude that a decoupling of nest predator abundance and nest predation may create ecological traps in human-modified environments.     

Genome-Wide Study of Structural Variants in Bovine Holstein,Montbéliarde and Normande Dairy Breeds

High-throughput sequencing technologies have offered in recent years new opportunities to study genome variations. These studies have mostly focused on single nucleotide polymorphisms, small insertions or deletions and on copy number variants. Other structural variants, such as large insertions or deletions, tandem duplications, translocations, and inversions are less well-studied, despite that some have an important impact on phenotypes. In the present study, we performed a large-scale survey of structural variants in cattle. We report the identification of 6,426 putative structural variants in cattle extracted from whole-genome sequence data of 62 bulls representing the three major French dairy breeds. These genomic variants affect DNA segments greater than 50 base pairs and correspond to deletions, inversions and tandem duplications. Out of these, we identified a total of 547 deletions and 410 tandem duplications which could potentially code for CNVs. Experimental validation was carried out on 331 structural variants using a novel high-throughput genotyping method. Out of these, 255 structural variants (77%) generated good quality genotypes and 191 (75%) of them were validated. Gene content analyses in structural variant regions revealed 941 large deletions removing completely one or several genes, including 10 single-copy genes. In addition, some of the structural variants are located within quantitative trait loci for dairy traits. This study is a pan-genome assessment of genomic variations in cattle and may provide a new glimpse into the bovine genome architecture. Our results may also help to study the effects of structural variants on gene expression and consequently their effect on certain phenotypes of interest.