Antifibrotic effect of the Chinese herbs, Astragalus mongholicus and Angelica sinensis, in a rat model of chronic puromycin aminonucleoside nephrosis

Nephrotic syndrome has long been treated in China with two herbs, Astragalus mongholicus and Angelica sinensis, which may have antifibrotic effects. METHODS: Rats with chronic puromycin-induced nephrosis were treated with Astragalus and Angelica 3 mL/d (n = 7) or enalapril 10 mg/kg/d (n = 7). Normal control rats (n = 7) received saline rather than puromycin, and an untreated control group (n = 7) received puromycin but no treatment. After 12 weeks, stained sections of the glomerulus and tubulointerstitium were evaluated for injury. Immunohistochemistry staining measured extracellular matrix components, transforming growth factor-beta1 (TGFbeta1), osteopontin, ED-1-positive cells, and alpha-actin. TGFbeta1 mRNA was assessed by in situ hybridization. Renin, ACE activity, angiotensin, and aldosterone were measured by radioimmunoassay or colorimetry. In the untreated rats, chronic renal injury progressed to marked fibrosis at 12 weeks. Astragalus and Angelica significantly reduced deterioration of renal function and histologic damage. Expressions of type III and IV collagen, fibronectin, and laminin also decreased significantly. This anti-fibrotic effect was similar to that of enalapril. The herbs had no effect on the renin-angiotensin system but did reduce the number of ED-1-positive, and alpha-actin positive cells and expression of osteopontin compared to untreated controls. The combination of Astragalus and Angelica retarded the progression of renal fibrosis and deterioration of renal function with comparable effects of enalapril. These effects were not caused by blocking the intrarenal renin-angiotensin system, but associated with suppression of the overexpression of TGFbeta1 and osteopontin, reduction of infiltrating macrophages, and less activation of renal intrinsic cells [corrected].     

Molecular phylogenetic relationships of China Seas groupers based on cytochrome <Emphasis Type="Italic">b</Emphasis> gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and T _S/T _V ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91(phox -/-) (X-linked) and p47 (phox -/-) (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.     

Allosteric inhibitors of Bcr-abl-dependent cell proliferation

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-abl, a 210-kDa fusion protein with deregulated tyrosine kinase activity. Encouraged by the clinical validation of Bcr-abl as the target for the treatment of CML by imatinib, we sought to identify pharmacological agents that could target this kinase by a distinct mechanism. We report the discovery of a new class of Bcr-abl inhibitors using an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library. Compounds in this class (exemplified by GNF-2) show exclusive antiproliferative activity toward Bcr-abl-transformed cells, with potencies similar to imatinib, while showing no inhibition of the kinase activity of full-length or catalytic domain of c-abl. We propose that this new class of compounds inhibits Bcr-abl kinase activity through an allosteric non-ATP competitive mechanism.     

Chemical fingerprint analysis of rhizomes of Gymnadenia conopsea by HPLC-DAD-MSn

A high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS(n)) method has been firstly developed for chemical fingerprint analysis of rhizomes of Gymnadenia conopsea R. Br. and rapid identification of major compounds in the fingerprints. Comparing the UV and MS spectra with those of reference compounds, seven main peaks in the fingerprints were identified as adenosine (1), 4-hydroxybenzyl alcohol (2), 4-hydroxybenzyl aldehyde (3), dactylorhin B (4), loroglossin (5), dactylorhin A (6) and militarine (7). Compounds 4-7 were succinate derivative esters and firstly discovered from this species. The Computer Aided Similarity Evaluation System (CASES) for chromatographic fingerprint of traditional Chinese medicine was employed to evaluate the similarities of 10 samples of the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provinces, Tibet autonomous region of China, and Nepal. These samples from different sources had similar chemical fingerprints. This method is specific and may serve for quality identification and comprehensive evaluation of this traditional Tibetan remedy.     

Sensitive HPLC-APCI-MS method for the determination of cyclovirobuxine D in human plasma

A sensitive high performance liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (HPLC-APCI-MS) assay for determination of cyclovirobuxine D (CVB-D) in human plasma using mirtazapine as internal standard (I.S.) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C(18) column with a mobile phase of 30 mM ammonium acetate buffer solution containing 1% formic acid-methanol (48:52, v/v). CVB-D was determined with atmospheric pressure chemical ionisation-mass spectrometry (APCI-MS). HPLC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at [M+H](+)m/z 403.4 for CVB-D and [M+H](+)m/z 266.2 for I.S. Calibration curves were linear over the range 10.11-4044 pg/ml. The lower limit of quantification was 10.11 pg/ml. The intra- and inter-run variability values were less than 9.5 and 12.4%, respectively. The mean plasma extraction recovery of CVB-D was in the range of 85.3-92.8%. The method was successfully applied to determine the plasma concentrations of CVB-D in Chinese volunteers.     

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.