Combinatorial mutation on the -glycosidase specific to 7--xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

Taxol is a "blockbuster" antitumor drug produced by species with extremely low amount, while its analogue 7--xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1-1 and LXYL-P1-2 can convert 7--xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1-2 is twice more active than LXYL-P1-1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1-1 to LXYL-P1-2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1-2 on -xylosidase and -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7--xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant , the molecular chaperone gene and the bacterial hemoglobin gene . This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1-2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1-2 for industrial purpose.

An improved liquid nitrogen freezing method for establishing canine models of femoral head necrosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an canine model of femoral head osteonecrosis using an improved liquid nitrogen freezing method.</p><p><b>METHODS</b>Sixteen adult canines were divided into 4 groups at random and subjected to instant freezing of the unilateral femoral head with liquid nitrogen. The dogs were observed at 2, 4, 8, and 16 weeks after the operation for radiographic, gross and pathological changes of the femoral head.</p><p><b>RESULTS</b>Significant radiographic, gross and pathological changes occurred in the femoral head after the freezing.</p><p><b>CONCLUSION</b>Improved liquid nitrogen freezing of the femoral head provides a simple and convenient method for establishing animal models of femoral head osteonecrosis.</p>

Expressions of c-Fos and NADPH-d in the related brainstem during vestibular compensation / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the mechanism of vestibular compensation and to observe the changes of c-Fos and NADPH-d expressions in the brainstem of the vestibular deafferentation rats in static status or following angular acceleration stimulation.</p><p><b>METHODS</b>Totally 60 SD rats were randomly divided into control group (labyrinthine intact), complete unilateral vestibular deafferentation (UVD) group, simultaneous complete bilateral vestibular deafferentation (BVD) group (n = 20 in each group). Subgroups (n = 10 in each subgroup) were set for static status or following angular acceleration stimulation in each group. Double labeling with histochemistry-immunohistochemistry was performed to observe c-Fos/NADPH-d neurons.</p><p><b>RESULTS</b>No positive c-Fos/NADPH-d expression was observed in the both sides of medial vestibular nucleus (MVN) and prepositus hypoglossi (PrH) of normal rats in static status and BVD rats whether following canal rotation or not. c-Fos/ NADPH-d expression was observed in the ipsilesional MVN and the contralesional PrH of UVD rats. However, c-Fos/NADPH-d were detected in both sides of MVN and PrH in UVD rats and normal rats following angular acceleration stimulation.</p><p><b>CONCLUSION</b>In the ipsilesional MVN and the contralesional PrH, c-Fos plays an important role in vestibular compensation, in which nitric oxide acts as a key neurotransmitter.</p>

Clinical analysis of normal subjects and dizzy patients with postural sway test / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the characteristics of the normal subjects with postural sway test and discuss the value of postural sway test in the diagnosis of dizzy patients.</p><p><b>METHODS</b>Totally 112 normal subjects, 72 patients with peripheral vertigo, and 30 patients with central vertigo were examined using a stabilometer (EAB-100, Anima Co., Japan). Items include patterns, length of locus, envelope area, deflection average center displacement, and romberg rate were recorded and compared.</p><p><b>RESULTS</b>The postural sway was classified into the following five patterns: centripetal, forward and backward, right and left, diffuse, and multicentric. Centripetal pattern was the most prominent figure pattern in the normal subjects, while diffuse pattern was the most prominent figure pattern in the patients with central vertigo. When the normal subjects closed their eyes, age was linearly correlated with the length of locus, envelope area, rectangel area, and unit area path length (P < 0.05). Sex was not correlated to all of these parameters. The length of locus and envelope area in the patient groups were significantly increased when compared with the normal subjects (P < 0.0167). Romberg rate was not significantly different between the patients and the normal subjects.</p><p><b>CONCLUSIONS</b>The pattern of postural sway is useful for the diagnosis of vertigo. Age is an important factor that affects the balance function. Sex seems has no influence. Visual input plays an important role in maintaining postural balance. Stabilometer is a useful tool for evaluating balance function, and postural sway test may be a valuable assistant examination.</p>

Establishment and characteristics of a human chordoma cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To present an established human chordoma cell line for chordoma research.</p><p><b>METHODS</b>The specimens pathologically identified as chordoma were cultured, using primary tissue culture in vitro. The surviving cells were analyzed by morphology, histochemical stain, cell cycling analysis, karyotype analysis, electron microscopic observation, heterotransplantation and study of invasive capacity in vitro.</p><p><b>RESULTS</b>The newly established cell line CM-319 has been maintained in continual cultures for over 100 generations in two years. Its morphological observation, histochemical staining properties, electron microscopic observation and heterotransplantation showed the common characteristics of chordoma. The doubling time of cells was about 33 hours. Cell cycle analysis showed: G(1) 55.6%, G(2) 21.9% and S 22.5%, G(2)/G(1) = 1.90. Chromosome analysis showed a hypotriploid feature and the success rate of heterotransplantation was 100%. It is capable of invasion in vitro.</p><p><b>CONCLUSION</b>CM-319, as a cell line derived from human chordoma cells, may serve for further studies of chordoma.</p>