Crystallization and X-ray analysis of NH3-dependent NAD+ synthetase from Helicobacter pylori

The ubiquitous NAD(+) synthetase catalyzes the key step in the biosynthesis of nicotinamide adenine dinucleotide. NH3-dependent NAD(+) synthetase from Helicobacter pylori was purified to homogeneity and crystallized using PEG 1500 as a precipitant. The crystal diffracted up to a resolution of 2.3+ and was found to belong to space group C2 with unit cell dimensions of a = 93.8, b = 48.3, c = 64.2 A and alpha = gamma = 90, beta = 110.0 degrees.     

Abnormal Tau phosphorylation of the Alzheimer-type also occurs during mitosis

In Alzheimer's disease, neurofibrillary degeneration results from the aggregation of abnormally phosphorylated Tau proteins into filaments and it may be related to the reactivation of mitotic mechanisms. In order to investigate the link between Tau phosphorylation and mitosis, Xenopus laevis oocytes in which most of the M-phase regulators have been discovered were used as a cell model. The human Tau isoform htau412 (2+3-10+) was microinjected into prophase I oocytes that were then stimulated by progesterone that activate cyclin-dependent kinase pathways. Hyperphosphorylation of the Tau isoform, which is characterized by a decrease of its electrophoretic mobility and its labelling by a number of phosphorylation-dependent antibodies, was observed at the time of germinal vesicle breakdown. Surprisingly, Tau immunoreactivity, considered as typical of Alzheimer's pathology (AT100 and phospho-Ser422), was observed in meiosis II. Because meiosis II is considered as a mitosis-like phase, we investigated if our observation was also relevant to a neurone-like model. Abnormal Tau phosphorylation was detected in mitotic human neuroblastoma SY5Y cells overexpressing Tau. Regarding AT100-immunoreactivity and phospho-Ser422, we suggest that phosphatase 2A inhibition and a phosphorylation combination of mitotic kinases may lead to this Alzheimer-type phosphorylation. Our results not only demonstrate the involvement of mitotic kinases in Alzheimer-type Tau phosphorylation but also indicate that Xenopus oocyte could be a useful model to identify the kinases involved in this process.     

The generation of Th memory in neonates versus adults: prolonged primary Th2 effector function and impaired development of Th1 memory effector function in murine neonates

Immunization during the neonatal period often results in Th2-biased secondary responses. To understand the regulation of this phenomenon, we have examined all phases of Th development, from the generation of primary effectors to the duration of the primary effector stage to the production of memory effector function. First, we had previously reported that although primary responses in the neonatal lymph nodes are mature, mixed Th1/Th2-like, primary responses in the spleens of the same animals are exclusively Th2-like. To determine whether Th2-dominant secondary responses are due to the Th2-polarized primary function in the spleen, neonates were splenectomized before immunization. Even in the absence of primary neonatal splenic responses, the secondary responses of neonates were Th2 dominant. Thus, the overwhelmingly Th2 primary responses in the neonatal spleen are not required to generate Th2-dominant memory in the lymph nodes. Second, we have compared the kinetics of the primary response phase in neonates and adults. In adults, Ag-specific Th2 function disappeared rapidly from both the lymph nodes and spleen. In contrast, primary Th2 function persisted out to 5 wk in both neonatal organs. Third, the generation of Th memory responses was examined in animals initially immunized as neonates and in adults. These experiments demonstrated that neonates are selectively impaired in the development of Th1 memory effector function. Together, these results indicate that neonates are biased to Th2 function at all phases of an immune response.     

血红蛋白的化学修饰与血液代用品

血液代用品系指具有携氧扩容功效的溶液,包括：氟碳化合物、脂质体包封血红蛋白（hemoglobin,Hb)、微囊化血红蛋白和无细胞基质血红蛋白（化学修饰Ｈb、基因重组Ｈb、化学合成Ｈb和转基因动物人Ｈb）。通过化学修饰稳定血红蛋白结构以避免其肾毒性,并延长循环半寿期是利用无基质血红蛋白制备血液代用品的主要研究内容,通常采用交联、聚合、偶联等方法完成。     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

The Effect of C-Terminal Deletion on the Folding and Self-association of Recombinant Non-phosphorylated Human ß-Casein

Recombinant human ß-casein (CN) mutants were prepared having 11, 22 and 31 amino acids (aa) deleted from the C-terminus. The temperature-dependent self-association of these and the wild-type recombinant was studied by turbidity (OD₄₀₀) while possible folding differences were examined by intrinsic and extrinsic fluorescence intensity and fluorescence resonance energy transfer. There were major self-association and some conformational differences. Hydrophobicity profile and hydrophobic cluster analysis for bovine and human ß-CN suggested that the ability of the 31 aa deletion mutant in human ß-CN to self-associate when a comparable bovine deletion peptide would not may be due to the presence of additional hydrophobic regions in the middle, indicating that the human protein may contain more than a single hydrophobic binding locus and suggesting that the process for the formation and structure of the micelles of human milk may be quite different from that for bovine milk. A new model may be needed.     

Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy

Rh2 is a ginsenoside extracted from ginseng that has drawn attention in a few laboratories in Asian countries because of its potential tumor-inhibitory effect. In the present study, we tested Rh2 on many tumor-cell lines for its effects on cell proliferation, induction of apoptosis, and potential interaction with conventional chemotherapy agents. Our results showed that Rh2 inhibited cell growth by G1 arrest at low concentrations and induced apoptosis at high concentrations in a variety of tumor-cell lines, possibly through activation of caspases. The growth arrest and apoptosis may be mediated by 2 separate mechanisms. Apoptosis is not dependent on expression of the wild-type p53 nor the caspase 3. In addition, the apoptosis induced by Rh2 was mediated through glucocorticoid receptors. Most interestingly, Rh2 can act either additively or synergistically with chemotherapy drugs on cancer cells. Particularly, it hypersensitized multidrug-resistant breast cancer cells to paclitaxel. These results suggest that Rh2 possesses strong tumor-inhibiting properties, and potentially can be used in treatments for multidrug-resistant cancers, especially when it is used in combination with conventional chemotherapy agents.     

Independent and cooperative roles of N-glycans and molecular chaperones in the folding and disulfide bond formation of the low-density lipoprotein (LDL) receptor-related protein

The low-density lipoprotein receptor-related protein (LRP) is a large receptor that contains extensive glycosylation sites and disulfide bonds. Here we analyzed how N-linked glycosylation and molecular chaperones function during LRP folding. Treatment of cells with a glycosylation inhibitor tunicamycin significantly impaired LRP folding, although binding to receptor-associated protein (RAP), a specialized chaperone for LRP, was not affected. The effects of tunicamycin on LRP folding were not due to an inhibition of RAP glycosylation since a mutant RAP that harbors a mutation at its sole glycosylation site was still capable of promoting LRP folding. The roles of N-linked glycosylation and the lectin chaperone, calnexin, in LRP folding were further dissected using LRP minireceptors that carry mutations at individual glycosylation sites. Interestingly, we found that RAP interacts with oxidoreductase ERp57 and mediates its interaction with LRP. Since previous studies have shown that N-glycan-bound calnexin/calreticulin are also capable of recruiting ERp57, our results suggest that N-linked glycosylation and RAP can independently and cooperatively recruit oxidoreductases to facilitate protein folding and proper disulfide bond formation.