H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Photoprotection of residual functional photosystem II units that survive illumination in the absence of repair, and their critical role in subsequent recovery

Photosystem II (PSII) complexes, which split water into oxygen, protons and electrons in photosynthesis, require light but are also inactivated by it. Recovery of PSII from photoinactivation requires de novo protein synthesis. PSII in capsicum leaf segments were photoinactivated in the absence of chloroplast-encoded protein synthesis. At large photon exposures and despite the absence of repair, a residual fraction of PSII remained functional, being ca 0.08–0.2 depending on the ease of gas exchange in the tissue. This study revealed that the residual functional PSII was photoprotected by both (1) reaction-center quenching of excitation energy by photoinactivated PSII even when little or no PSII activity was permitted, and (2) antenna quenching, which was dependent on a trans-thylakoid pH gradient sustained mainly by linear electron transport and facilitated by the residual functional PSII complexes themselves. Significantly, little or no contribution to photoprotection of PSII was observed from cyclic electron flow around PSI. Further, the small residual functional PSII population was critical for recovery of the photoinactivated PSII complexes. Thus, photoinactivated and residual functional PSII complexes in leaves play a mutually beneficial role in each other's ultimate survival.     

An integrated map of Oryza sativa L. chromosome 5

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.     

Little population structuring and recent evolution of the Pacific saury (Cololabis saira) as indicated by mitochondrial and nuclear DNA sequence data

Genetic population structure of the Pacific saury (Cololabis saira) was investigated using nucleotide sequence analysis on the mitochondrial DNA control region (355-361 bp). Although the left domain of the control region is known to be highly variable in many species, extremely low nucleotide and haplotype diversities (π = 0.17% and h = 0.418, respectively) were observed in a total of 141 individuals collected from five distant locales (East China Sea, Sea of Okhotsk, northwest Pacific, central North Pacific and northeast Pacific). No significant haplotype frequency differences were detected among widely separated samples, therefore we were unable to reject the null hypothesis of no genetic structuring in the Pacific saury population. Moderate levels of nucleotide substitution (p-distance) were observed between the Pacific saury and its Atlantic counterpart (Scomberesox saurus) in the control region (7.44%), cytochrome b gene (4.64%), and internal transcribed spacer (ITS1) (11.49%), indicating that the low sequence diversity of the control region in the Pacific saury is not due to the slow mutation rate. The molecular data suggest the Pacific saury may be a relatively recent offshoot among the extant members in the family Scomberesocidae.     

Calmodulin Binds a Highly Extended HIV-1 MA Protein That Refolds Upon Its Release

Calmodulin (CaM) expression is upregulated upon HIV-1 infection and interacts with proteins involved in viral processing, including the multifunctional HIV-1 MA protein. We present here the results of studies utilizing small-angle neutron scattering with contrast variation that, when considered in the light of earlier fluorescence and NMR data, show CaM binds MA in an extended open-clamp conformation via interactions with two tryptophans that are widely spaced in sequence and space. The interaction requires a disruption of the MA tertiary fold such that MA becomes highly extended in a long snakelike conformation. The CaM-MA interface is extensive, covering ～70% of the length of the MA such that regions known to be important in MA interactions with critical binding partners would be impacted. The CaM conformation is semiextended and as such is distinct from the classical CaM-collapse about short α-helical targets. NMR data show that upon dissociation of the CaM-MA complex, either by the removal of Ca(2+) or increasing ionic strength, MA reforms its native tertiary contacts. Thus, we observe a high level of structural plasticity in MA that may facilitate regulation of its activities via intracellular Ca(2+)-signaling during viral processing.     

Facultative gregarious development in a solitary koinobiont parasitoid,Ephedrus californicus: implications of larval ecology and host constraints

In solitary parasitoids, the mandibulate first instars behave aggressively towards potential competitors so that generally only one larva survives per host. A ‘failure of competition’ may result in facultative gregarious development, however. We used Ephedrus californicus Baker (Hymenoptera: Braconidae: Aphidiinae), a solitary koinobiont parasitoid of aphids, to test two hypotheses in the laboratory that could explain facultative gregarious development. Gregarious development increased with the intensity of parasitism, with two (rarely three) parasitoids successfully developing in a single aphid. In heavily superparasitized hosts, interference between surviving larvae often caused abnormal pupation behaviour and inability to emerge from the mummy. The hypothesis that the survival of more than one larva per host is dependent on differences in larval age was not supported. The total body size in terms of dry mass of two males or two females developing together in the same host was higher than that of same‐sex counterparts developing singly. Females were larger than males with which they shared a host. Hypotheses about the evolutionary transition from a solitary to a gregarious lifestyle in parasitoid Hymenoptera have focused on lethal fighting between first instars but have ignored other constraints including immature mortality during later development and limiting host resources. Especially in species that pupate inside the dead host, specific requirements for pupation and emergence may determine whether one or several offspring per host can develop to adult.     

Comparison of solution conformations and stabilities of modified helix 69 rRNA analogs from bacteria and human

The helix 69 (H69) region of the large subunit (28S) ribosomal RNA (rRNA) of Homo sapiens contains five pseudouridine (Ψ) residues out of 19 total nucleotides, three of which are highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine in double-stranded (stem) regions. These results were compared with previous hairpin (stem plus single-stranded loop) studies to understand the contributions of the loop sequences to H69 structure and stability. The role of a loop nucleotide substitution from an A in bacteria (position 1918 in Escherichia coli 23S rRNA) to a G in eukaryotes (position 3734 in H. sapiens 28S rRNA) was examined. Thermodynamic parameters for the duplex RNAs were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by circular dichroism spectroscopy. The overall folded structure of human H69 appears to be similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved; however, our results reveal subtle differences in structure and stability between the bacterial and human H69 RNAs in both the stem and loop regions. These findings may be significant with respect to H69 as a potential drug target site.     

Specialized Hsp70 Chaperone (HscA) Binds Preferentially to the Disordered Form, whereas J-protein (HscB) Binds Preferentially to the Structured Form of the Iron-Sulfur Cluster Scaffold Protein (IscU)

The Escherichia coli protein IscU serves as the scaffold for Fe-S cluster assembly and the vehicle for Fe-S cluster transfer to acceptor proteins, such as apoferredoxin. IscU populates two conformational states in solution, a structured conformation (S) that resembles the conformation of the holoprotein IscU-[2Fe-2S] and a dynamically disordered conformation (D) that does not bind metal ions. NMR spectroscopic results presented here show that the specialized Hsp70 chaperone (HscA), alone or as the HscA-ADP complex, preferentially binds to and stabilizes the D-state of IscU. IscU is released when HscA binds ATP. By contrast, the J-protein HscB binds preferentially to the S-state of IscU. Consistent with these findings, we propose a mechanism in which cluster transfer is coupled to hydrolysis of ATP bound to HscA, conversion of IscU to the D-state, and release of HscB.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: MECHANISMS UNDERLYING GASTROINTESTINAL INCORPORATION OF THE NON-HUMAN SIALIC ACID XENO-AUTOANTIGEN N-GLYCOLYLNEURAMINIC ACID

Although N-acetyl groups are common in nature, N-glycolyl groups are rare. Mammals express two major sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). Although humans cannot produce Neu5Gc, it is detected in the epithelial lining of hollow organs, endothelial lining of the vasculature, fetal tissues, and carcinomas. This unexpected expression is hypothesized to result via metabolic incorporation of Neu5Gc from mammalian foods. This accumulation has relevance for diseases associated with such nutrients, via interaction with Neu5Gc-specific antibodies. Little is known about how ingested sialic acids in general and Neu5Gc in particular are metabolized in the gastrointestinal tract. We studied the gastrointestinal and systemic fate of Neu5Gc-containing glycoproteins (Neu5Gc-glycoproteins) or free Neu5Gc in the Neu5Gc-free Cmah(-/-) mouse model. Ingested free Neu5Gc showed rapid absorption into the circulation and urinary excretion. In contrast, ingestion of Neu5Gc-glycoproteins led to Neu5Gc incorporation into the small intestinal wall, appearance in circulation at a steady-state level for several hours, and metabolic incorporation into multiple peripheral tissue glycoproteins and glycolipids, thus conclusively proving that Neu5Gc can be metabolically incorporated from food. Feeding Neu5Gc-glycoproteins but not free Neu5Gc mimics the human condition, causing tissue incorporation into human-like sites in Cmah(-/-) fetal and adult tissues, as well as developing tumors. Thus, glycoproteins containing glycosidically linked Neu5Gc are the likely dietary source for human tissue accumulation, and not the free monosaccharide. This human-like model can be used to elucidate specific mechanisms of Neu5Gc delivery from the gut to tissues, as well as general mechanisms of metabolism of ingested sialic acids.