Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule.     

不同胰岛素给药方式救治糖尿病酮症酸中毒临床研究

目的：比较不同胰岛素给药方式治疗糖尿病酮症酸中毒（DKA）的临床疗效。方法：82例DKA患者随机分为胰岛素泵持续皮下输液胰岛素（CSⅡ）组和微量泵持续静脉泵入胰岛素（CXqI）组各41例,分别给予胰岛素泵持续皮下输注胰岛素和小剂量胰岛素持续微量泵静脉泵入不同胰岛素给药方式,观察两组治疗后血糖变化、血糖达标时间、尿酮体变化、pH值变化、胰岛素平均日用量、平均低血糖次数及平均住院时间。结果：两组治疗后空腹血糖、餐后血糖显著下降及血糖达标时间显著缩短差异无统计学意义（P〉0．05）;CSII组尿酮体转阴时间（22．3±7．4）h短于CVII组（32．1±12．1）h（P〈0．01）;CSII组PH值恢复时间（9．4±2．5）h短于CVII组（15．7±3．5）h（P〈0．01）;CSII组平均胰岛素日用量为（47±5）U比CVII组（58＋7）U少（P〈0．01）;CSII组人均低血糖次数为（0．6±O．5）次／人。少于CVII组（1．5±0．8）次／人（P〈O．01）;CSII组住院时间（9．8±1．2）天明显比CVII组（12．5±2．0）天短（P〈0．01）。结论：CSII相较于CVII能更快更有效的纠正代谢紊乱,减少胰岛素日用量,缩短住院时间,从而提高临床疗效。具有较高的安全性及患者依从性。     

Cloning and sequencing of the gerD gene of Bacillus subtilis

A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsI. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnI. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1.8 kb fragment of DNA which complemented ger-97::Tn917 and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential sigma E and sigma A promoter regions and encoding a predicted polypeptide of 21,117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.     

Cell therapy for skin wound using fibroblast encapsulated poly(ethylene glycol)-poly(L-alanine) thermogel

As a new application of a thermogel, a poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with Matrigel for cell proliferation and was significantly better than Matrigel for collagen types I and III formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the cell-encapsulated gel was formed in situ. Compared with the phosphate buffered saline treated system and the cell-free PEG-L-PA thermogel, the cell-encapsulated PEG-L-PA thermogel not only accelerated the wound closure but also improved epithelialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. The results demonstrate the potential of thermogels for cell therapy as an injectable tissue-engineering scaffold.     

PAC1 receptor activation by PACAP-38 mediates Ca2+ release from a cAMP-dependent pool in human fetal adrenal gland chromaffin cells

Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.     

PTP-1B inhibitors: cyclopenta[d][1,2]-oxazine derivatives

A series of novel cyclopenta[d][1,2]-oxazine derivatives was prepared and evaluated for their inhibitory activity toward protein tyrosine phosphatase 1B (PTP-1B). Compound 6s was found to be an inhibitor of PTP-1B with nanomolar IC(50) value and high level of selectivity over other recombinant phosphatases.