Tracking the immunoregulatory mechanisms active during allograft tolerance

Immunoregulatory mechanisms dependent on regulatory CD4+ T cells are believed to be critical in the maintenance of peripheral tolerance to allografts. However, a detailed characterization of the effects of these regulatory T cells has been hampered by the absence of a simple means to track and study them. In this work we provide evidence that in a murine model of islet transplantation the interactions between alloaggressive and regulatory T cells can be studied in vitro and in vivo at the single-cell level. The observations made in both an in vitro coculture system and an in vivo CFSE-based adoptive transfer model indicate that lymphocytes from tolerant allograft recipients 1) proliferate weakly to donor strain allogeneic cells but vigorously to third-party strain cells; and 2) suppress the proliferation of naive syngeneic CD4+ and CD8+ T cells to donor tissue in a cell dose- and Ag-specific manner. These effects depend on the presence of CD4+CD25+ T cells and are neutralized by anti-CTLA4 mAb or rIL-2. The principal effect of anti-CTLA4 is directed against the naive, not regulatory, T cell population. These results can be replicated in vivo by transferring lymphocyte populations into transplant recipients, proving that the graft-protecting actions of regulatory T cells are blunted by a rise in the number of allodestructive T cells (pool size model) and depend on the presence of CD4+CD25+ T cells and the integrity of the CTLA4/B7 pathway.     

Ubiquitin-fused and/or multiple early genes from cottontail rabbit papillomavirus as DNA vaccines

Human papillomavirus (HPV) vaccines have the potential to prevent cervical cancer by preventing HPV infection or treating premalignant disease. We previously showed that DNA vaccination with the cottontail rabbit papillomavirus (CRPV) E6 gene induced partial protection against CRPV challenge and that the vaccine's effects were greatly enhanced by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, two additional strategies for augmenting the clinical efficacy of CRPV E6 vaccination were evaluated. The first was to fuse a ubiquitin monomer to the CRPV E6 protein to enhance antigen processing and presentation through the major histocompatibility complex class I pathway. Rabbits vaccinated with the wild-type E6 gene plus GM-CSF or with the ubiquitin-fused E6 gene formed significantly fewer papillomas than the controls. The papillomas also required a longer time to appear and grew more slowly. Finally, a significant proportion of the papillomas subsequently regressed. The ubiquitin-fused E6 vaccine was significantly more effective than the wild-type E6 vaccine plus GM-CSF priming. The second strategy was to vaccinate with multiple CRPV early genes to increase the breadth of the CRPV-specific response. DNA vaccines encoding the wild-type CRPV E1-E2, E6, or E7 protein were tested alone and in all possible combinations. All vaccines and combinations suppressed papilloma formation, slowed papilloma growth, and stimulated subsequent papilloma regression. Finally, the two strategies were merged and a combination DNA vaccine containing ubiquitin-fused versions of the CRPV E1, E2, and E7 genes was tested. This last vaccine prevented papilloma formation at all challenge sites in all rabbits, demonstrating complete protection.     

A relative value method for measuring and evaluating cardiac reserve

Background  

Although a very close relationship between the amplitude of the first heart sound (S1) and the cardiac contractility have been proven by previous studies, the absolute value of S1 can not be applied for evaluating cardiac contractility. However, we were able to devise some indicators with relative values for evaluating cardiac function.     

NMR imaging of high-amylose starch tablets. 2. Effect of tablet size

Carbohydrate polymers are widely used for pharmaceutical applications such as the controlled release of drugs. The swelling and water mobility in high-amylose starch tablets are important parameters to be determined for these applications. They have been studied at different time intervals by nuclear magnetic resonance imaging (NMRI) after the immersion of the samples in water. These tablets have a hydrophilic matrix, which swells anisotropically and forms a hydrogel in water. NMRI shows clearly the anisotropy of the water penetration and the swelling along the radial and axial dimensions of the tablets. Empirical relationships are established to describe the kinetics of water penetration and swelling of the tablets. Results show that water uptake and tablet swelling strongly depend on the size of the tablets. Gravimetric measurements of water uptake were also performed in comparison with the NMRI results.     

Induction of cardiac cytochrome p450 in cocaine-treated mice

Cytochrome P450 (P450) is a ubiquitous family of enzymes responsible for the metabolism of a wide variety of drugs and their metabolites, including cocaine. To investigate the effects of cocaine on myocardial injuries and cardiac P450 expression, BALB/c mice were injected daily intraperitoneally with cocaine (30 mg/kg) or cocaine plus pretreatment of P450 inhibitors for 14 days. Tumor necrosis factor-alpha (TNF-alpha) content and creatine phosphokinase (CPK) activity in mice hearts and serums were significantly increased after long-term treatment with cocaine. Pretreatment with the P450 inhibitor, cimetidine (Cime, 50 mg/kg) or metyrapone (Mety, 40 mg/kg) abolished or significantly attenuated the effects of cocaine on TNF-alpha and CPK activity. Western blot analysis shows that mouse cardiac tissues express the P450 isoforms CYP1A1, CYP1A2, and CYP2J2. The protein levels normalized with cyclophilin A were 1.20 plus minus 0.07, 0.67 plus minus 0.03, and 1.48 plus minus 0.01 for CYP1A1, CYP1A2, and CYP 2J2, respectively. After cocaine administration, CYP2J2 increased by 43.6% and CYP1A1 increased by 108.5%, but CYP1A2 was not significantly altered. However, the cytochrome P450 inhibitors Cime and Mety suppressed the cocaine-induced increase in CYP1A1 and CYP2J2 expression. Moreover, application of Cime or Mety alone did not alter the level of cardiac TNF-alpha or the expression of P450. Our results demonstrate that long-term exposure to cocaine causes an increase in cardiac CYP1A1 and CYP2J2 concentration. We speculate that induction of P450 isoforms may cause cardiac injury due to cocaine metabolites locally catalyzed by P450 or the increase in P450 expression itself.     

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.     

An active precursor in assembly of yeast nuclear ribonuclease P

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.     

间种蔬菜对甘蔗地生态环境和甘蔗生长的影响

甘蔗 (ＳａｃｃｈａｒｕｍｏｆｆｉｃｉｎａｒｕｍＬ .)是广西重要的经济作物 ,种植面积达 4 5 33× 10 6ｈａ[1] ,甘蔗的前期生长比较缓慢 ,行间有比较大的可利用空间 ,到6月下旬～ 7月上旬才进入迅速生长期并逐渐封行。有研究表明 ,在甘蔗园间种玉米对甘蔗的生长发育和产量有一定的影响[5] ,而合理间种豆科作物 (黄豆和花生 )不仅不影响甘蔗的正常生长发育 ,还可以增加甘蔗的产量[4 ,6] 。但间种作物对甘蔗园生态环境的影响未见报道 ,我们于 1997- 1999年在桂西北开展了甘蔗园间种经济效益比较高的蔬菜作物的试验 ,并对间种甘蔗园生态环…     

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.