Allograft rejection by primed/memory CD8+ T cells is CD154 blockade resistant: therapeutic implications for sensitized transplant recipients

We have shown that CD8(+) CTLs are the key mediators of accelerated rejection, and that CD8(+) T cells represent the prime targets of CD154 blockade in sensitized mouse recipients of cardiac allografts. However, the current protocols require CD154 blockade at the time of sensitization, whereas delayed treatment fails to affect graft rejection in sensitized recipients. To elucidate the mechanisms of costimulation blockade-resistant rejection and to improve the efficacy of CD154-targeted therapy, we found that alloreactive CD8(+) T cells were activated despite the CD154 blockade in sensitized hosts. Comparative CD8 T cell activation study in naive vs primed hosts has shown that although both naive and primed/memory CD8(+) T cells relied on the CD28 costimulation for their activation, only naive, not primed/memory, CD8(+) T cells depend on CD154 signaling to differentiate into CTL effector cells. Adjunctive therapy was designed accordingly to deplete primed/memory CD8(+) T cells before the CD154 blockade. Indeed, unlike anti-CD154 monotherapy, transient depletion of CD8(+) T cells around the time of cardiac engraftment significantly improved the efficacy of delayed CD154 blockade in sensitized hosts. Hence, this report provides evidence for 1) differential requirement of CD154 costimulation signals for naive vs primed/memory CD8(+) T cells, and 2) successful treatment of clinically relevant sensitized recipients to achieve stable long term graft acceptance.     

CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.     

Requirement of spermidine for developmental transitions in Aspergillus nidulans

Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, deltaspdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when deltaspdA is grown on media supplemented with 0.05-0.10 mM exogenous spermidine. Supplementation of deltaspdA with >/= 0.10 mM spermidine restores sterigmatocystin production and >/= 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. DeltaspdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in deltaspdA strains grown in media lacking spermidine but could force both processes in deltaspdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition.     

Detecting single molecules launched from liquid surfaces in a time-of-flight mass spectrometer using ultraviolet and infrared lasers

The launch of molecules from liquid surfaces in a time of flight mass spectrometer has been investigated using different sample preparation techniques, and by exposing the liquid samples to two different laser wavelengths, 337 nm from a N2 ultraviolet laser and 10.6 microm from a CO2 infrared laser. The molecules were detected with cryodetectors measuring the energy of the individual molecules. We present insulin and lysozyme results from samples introduced into the vacuum through a micromachined silicon injector, and from samples consisting of a glycerol droplet deposited directly on the sample holder at the high voltage stage of the ion optics.     

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

MOTIVATION: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. RESULTS: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets. AVAILABILITY: See http://microarray.imb.uq.edu.au/golgi/     

ACh受体在皮质酮引起的大鼠延髓腹外侧区前交感神经元反应中的作用

目的：研究乙酰胆碱（ACh)受体在皮质酮（CORT）对大鼠头端延髓腹外侧区（RVLM）前交感神经元快速效应中的作用,探讨糖皮质激素在交感心血管活动调节中的非基因组机制。方法：本研究采用细胞外记录和微电泳等方法观察CORT对氨基甲酸乙酯麻醉大鼠RVLM前交感神经元的作用,观察分别给予ACh受体拮抗剂阿托品（ATR）、筒箭毒（d-TC)或六烃季铵（C6）后CORT对RVLM前交感神经元的影响。结果：在RVLM共记录到33个前交感神经元,CORT能导致25（76％）个前交感神经元快速兴奋,且具有剂量依赖性,余8个前交感神经元没有反应;其中被CORT兴奋的10个单位微电泳ART后神经元的放电明显下降,但对CORT导致的兴奋作用没有明显的影响。分别向7和6个被CORT兴奋的前交感神经元微电泳d-TC和C6后,单位放电没有变化,同时对CORT导致的兴奋作用无影响。结论：CORT对RVLM前交感神经元具有快速的兴奋作用,这种作用可能并不通过ACh受体介导。     

Construction of alpha-alpha domains structure in recombinant monkey metallothionein-1

The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.     

Astrocyte-specific inactivation of the neurofibromatosis 1 gene (NF1) is insufficient for astrocytoma formation

Individuals with the neurofibromatosis 1 (NF1) inherited tumor syndrome develop low-grade gliomas (astrocytomas) at an increased frequency, suggesting that the NF1 gene is a critical growth regulator for astrocytes. In an effort to determine the contribution of the NF1 gene product, neurofibromin, to astrocyte growth regulation and NF1-associated astrocytoma formation, we generated astrocyte-specific Nf1 conditional knockout mice (Nf1(GFAP)CKO) by using Cre/LoxP technology. Transgenic mice were developed in which Cre recombinase was specifically expressed in astrocytes by embryonic day 14.5. Successive intercrossing with mice bearing a conditional Nf1 allele (Nf1flox) resulted in GFAP-Cre Nf1flox/flox (Nf1(GFAP)CKO) animals. No astrocytoma formation or neurological impairment was observed in Nf1(GFAP)CKO mice after 20 months, but increased numbers of proliferating astrocytes were observed in several brain regions. To determine the consequence of Nf1 inactivation at different developmental times, the growth properties of embryonic day 12.5 and postnatal day 2 Nf1 null astrocytes were analyzed. Nf1 null astrocytes exhibited increased proliferation but lacked tumorigenic properties in vitro and did not form tumors when injected into immunocompromised mouse brains in vivo. Collectively, our results suggest that loss of neurofibromin is not sufficient for astrocytoma formation in mice and that other genetic or environmental factors might influence NF1-associated glioma tumorigenesis.     

Comparison of Some Photosynthetic Characters Between Two Hybrid Rice Combinations Differing in Yield Potential

Photosynthetic characteristics of two hybrid rice combinations, Peiai 64S/E32 and Shanyou 63, were compared at the panicle differentiation stage. As compared with Shanyou 63, the new combination Peiai 64S/E32 showed a significantly higher net photosynthetic rate (P _N), apparent quantum yield of carbon assimilation (_c), carboxylation efficiency (CE), and photorespiratory rate (R _P) as well as leaf chlorophyll content, but a significantly lower dark respiration rate (R _D) and compensation irradiance (I _c). It also showed a slightly higher photochemical efficiency (F_v/F_m and F/F_m) of photosystem 2, a lower non-photochemical quenching (q_N), and a similar CO₂ compensation concentration () as compared to Shanyou 63.