Combined abdominal heterotopic heart and aorta transplant model in mice.

BACKGROUND: Allograft vasculopathy (AV) remains a major obstacle to long-term allograft survival. While the mouse aortic transplantation model has been proven as a useful tool for study of the pathogenesis of AV, simultaneous transplantation of the aorta alongside the transplantation of another organ may reveal more clinically relevant mechanisms that contribute to the pathogenesis of chronic allograft rejection. Therefore, we developed a combined abdominal heart and aorta transplantation model in mice which benefits from reducing animal and drug utilization, while providing an improved model to study the progressive nature of AV. METHODS: The middle of the infrarenal aorta of the recipient mouse was ligatured between the renal artery and its bifurcation. Proximal and distal aortotomies were performed at this site above and below the ligature, respectively, for the subsequent anastomoses of the donor aorta and heart grafts to the recipient infrarenal aorta in an end-to-side fashion. The distal anastomotic site of the recipient infrarenal aorta was connected with the outlet of the donor aorta. Uniquely, the proximal anastomotic site on the recipient infrarenal aorta was shared to connect with both the inlet of the donor aorta and the inflow tract to the donor heart. The outflow tract from the donor heart was connected to the recipient inferior vena cava (IVC). RESULTS: The median times for harvesting the heart graft, aorta graft, recipient preparation and anastomosis were 11.5, 8.0, 9.0 and 40.5 min, respectively, resulting in a total median ischemic time of 70 min. The surgery survival rate was more than 96% (29/30). Both the syngeneic C57Bl/6 aorta and heart grafts survived more than 90 days in 29 C57Bl/6 recipients. Further, Balb/c to C57Bl/6 allografts treated with anti-CD40L and CTLA4.Ig survived more than 90 days with a 100% (3/3) survival rate. (3/3). CONCLUSIONS: This model is presented as a new tool for researchers to investigate transplant immunology and assess immunosuppressive strategies. It is possible to share a common anastomotic stoma on the recipient abdominal aorta to reconstruct both the aorta graft entrance and heart graft inflow tract. This allows for the study of allogeneic effects on both the aorta and heart from the same animal in a single survival surgery.

Molecular Determinants of Enterotoxigenic Escherichia coli Heat-Stable Toxin Secretion and Delivery.

Enterotoxigenic Escherichia coli (ETEC), a heterogeneous diarrheal pathovar defined by production of heat-labile (LT) and/or heat-stable (ST) toxins, causes substantial morbidity among young children in the developing world. Studies demonstrating a major burden of ST-producing ETEC have focused interest on ST toxoids for ETEC vaccines. We examined fundamental aspects of ST biology using ETEC strain H10407, which carries estH and estP genes encoding STh and STp, respectively, in addition to eltAB genes responsible for LT. Here, we found that deletion of estH significantly diminished cyclic GMP (cGMP) activation in target epithelia, while deletion of estP had a surprisingly modest impact, and a dual estH estP mutant was not appreciably different from the estH mutant. However, we noted that either STh or STp recombinant peptides stimulated cGMP production and that the loss of estP was compensated by enhanced estH transcription. We also found that the TolC efflux protein was essential for toxin secretion and delivery, providing a potential avenue for efflux inhibitors in treatment of acute diarrheal illness. In addition, we demonstrated that the EtpA adhesin is required for optimal delivery of ST and that antibodies against either the adhesin or STh significantly impaired toxin delivery and cGMP activation in target T84 cells. Finally, we used FLAG epitope fusions to demonstrate that the STh propeptide sequence is secreted by ETEC, potentially providing additional epitopes for antibody neutralization. These studies collectively extend our understanding of ETEC pathogenesis and potentially inform additional avenues to mitigate disease by these common diarrheal pathogens.

A Novel Redoxin in the Thylakoid Membrane Regulates the Titer of Photosystem I.

In photosynthetic organisms like cyanobacteria and plants, the main engines of oxygenic photosynthesis are the pigment-protein complexes photosystem I (PSI) and photosystem II (PSII) located in the thylakoid membrane. In the cyanobacterium Synechocystis sp. PCC 6803, the slr1796 gene encodes a single cysteine thioredoxin-like protein, orthologs of which are found in multiple cyanobacterial strains as well as chloroplasts of higher plants. Targeted inactivation of slr1796 in Synechocystis 6803 resulted in compromised photoautotrophic growth. The mutant displayed decreased chlorophyll a content. These changes correlated with a decrease in the PSI titer of the mutant cells, whereas the PSII content was unaffected. In the mutant, the transcript levels of genes for PSI structural and accessory proteins remained unaffected, whereas the levels of PSI structural proteins were severely diminished, indicating that Slr1796 acts at a posttranscriptional level. Biochemical analysis indicated that Slr1796 is an integral thylakoid membrane protein. We conclude that Slr1796 is a novel regulatory factor that modulates PSI titer.

Two new lignans from the stems of Schisandra bicolor.

Two new lignans, named zuihonins E (1) and F (2), were isolated from the stems of Schisandra bicolor Cheng var. tuberculata Law. The structures of the new lignans were elucidated on the basis of extensive spectroscopic analysis, including 1D, 2D NMR, and MS experiments, and their absolute stereochemistry was determined by circular dichroism spectrum. Compounds 1 and 2 did not inhibit the growth of hepatoma carcinoma cell (HepG2), lung carcinoma cell (A549), and human breast carcinoma (MCF-7) cell lines.

Characterization of cDNA of lycopene beta-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina.

Lycopene beta-cyclase (Lyc-B) is the key enzyme in the catalysis of linear lycopene to form cyclic beta-carotene, an indispensable part of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Studies showing that the microalga Dunaliella salina can accumulate a high level of beta-carotene are lacking. We hypothesize that D. salina is closely involved with the catalytic mechanism of Lyc-B and the molecular regulation of its gene. In this study, we used RT-PCR and RACE-PCR to isolate a 2475 bp cDNA with a 1824 bp open reading frame, encoding a putative Lyc-B, from D. salina. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with sequences of other green algae and higher plants, and that it shared the highest sequence identity, up to 64%, with Lyc-B of Chlamydomonas reinhardtii. Codon analysis showed that synonymous codon usage in the enzyme has a strong bias towards codons ending with adenosine. Two motifs were found in the Lyc-B sequence, one at the N terminus, for binding the hypothetical cofactor FAD, and the other was a substrate carrier motif in oxygenic organisms shared by an earlier carotenogenesis enzyme, phytoene desaturase, and Lyc-B. A tertiary structure prediction suggested that the catalytic or binding site structure within LycB from D. salina is superior to that of both H. pluvialis and C. reinhardtii. The LycB protein from D. salina was quite removed from that of H. pluvialis and C. reinhardtii in the phylogenetic tree. Taken as a whole, this information provides insight into the regulatatory mechanism of Lyc-B at the molecular level and the high level of beta-carotene accumulation in the microalga D. salina.