Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing–decreasing–increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.KEY WORDS: Glycyrrhiza uralensis Fisch., 3-Hydroxy-3-methylglutaryl-CoA reductase gene, Over-expression, Pichia pastoris, Copy number variationAbbreviations: BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; CNV, copy number variation; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; LOD, limit of detection; LLOQ, lower limit of quantitation; MD, minimal dextrose medium; MM, minimal medium; MVA, mevalonic acid; PCR, polymerase chain reaction; RSD, relative standard deviation; YPD, yeast peptone dextrose medium     

Early inflammatory response of knee ligaments to prolotherapy in a rat model

Prolotherapy is an alternative injection‐based therapy for chronic musculoskeletal pain. Three different proliferants, D ‐glucose (dextrose), phenol‐glucose‐glycerine (P2G), and sodium morrhuate, used in prolotherapy are hypothesized to strengthen and reorganize chronically injured soft tissue and decrease pain through modulation of the inflammatory process. Our hypothesis is that commonly used prolotherapy solutions will induce inflammation (leukocyte and macrophage infiltration) in medial collateral ligaments (MCLs) compared to needlestick, saline injection, and no‐injection controls. MCLs of 84 Sprague‐ Dawley rats were injected one time at both the tibial and femoral insertions. Immunohistochemistry (IHC) was used to determine the inflammatory response at three locations (tibial and femoral insertions and midsubstance) 6, 24, and 72 h after dextrose injection compared to saline‐ and no‐injection controls and collagenase (positive control) (n = 4). qPCR was used to analyze gene expression 24 h postinjection (n = 4). Sodium morrhuate, P2G, and needlestick control were also investigated after 24 h (n = 4). In general, inflammation (CD43+, ED1+, and ED2+ cells) increased after prolotherapy injection compared to no‐injection control but did not increase consistently compared to saline and needlestick control injections. This response varied by both location and proliferant. Inflammation was observed at 6 and 24 h postinjection but was resolved by 72 h compared to no‐injection controls (p < 0.05). CD43+ leukocytes and ED2+ macrophages increased compared to needlestick and saline‐injection control, respectively, 24 h postinjection (p < 0.05). Prolotherapy injections created an inflammatory response, but this response was variable and overall, not uniformly different from that caused by saline injections or needlestick procedures. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:816–823, 2008     

Transport Refusal by Hypoglycemic Patients after On-scene Intravenous Dextrose

OBJECTIVES: Administration of intravenous (IV) dextrose to hypoglycemic patients is delegated to advanced care paramedics in Ontario. Following a quality assurance review, which revealed that 47% of patients refused transport after receiving IV dextrose, the authors studied whether such patients seek additional medical care in the three days following the initial refusal. METHODS: Sequential ambulance call reports for on-scene treatments of hypoglycemia were examined, and a standardized telephone survey of the patients was conducted. Patient satisfaction was assessed using a five-point Likert scale. Data were collected from April 1999 to March 2000. RESULTS: One hundred patients were studied, with ages ranging from 20 to 92 years (mean 53.2 years). The average Glasgow Coma Scale (GCS) score on presentation was 8.7 +/- 3.5. The average blood glucose level before administration of IV dextrose was 1.91 +/- 0.63 mmol/L. Sixty-eight percent of the patients refused transport. Significant differences between the transported group and the refusal group were age (transported 64.7 years, refused 47.8 years, p = 0.002) and initial blood glucose (transported 1.8, refused 2.1, p = 0.001). No difference was found in terms of repeat access to health care for related complaints. Patient satisfaction was high in both groups, with no difference in the overall satisfaction with paramedics' care (4.76 +/- 0.58 vs 4.75 +/- 0.45). CONCLUSIONS: The practice of treating patients for symptomatic hypoglycemia and leaving them at the scene appears to be safe. Further study is required to confirm this.     

Storage of platelet concentrates harvested from blood collected into dextrose-free preservative without agitation

Summary. This study addresses the possibility of platelet quality being maintained during storage by an endogenous metabolic fuel, while avoiding dextrose-induced lactate accumulation. This was achieved by harvesting platelet concentrates from blood donations collected into a dextrose-free anticoagulant. Adequate maintenance of all metabolic and functional parameters was observed in platelets from blood collected into 4% citrate. The requirement for platelets stored in CPD plasma to be agitated during storage was confirmed, but agitation could be omitted for dextrose-free platelets without increased lactate generation and a drop in pH. These results indicate that platelet concentrates from dextrose-free blood may be stored without some of the constraints accompanying platelet storage in conventional media, and may thus result in improved delivery of this product.     

Variations of the phosphorylation of 1-β-d-arabinofuranosylcytosine (ara-C) in human myeloid leukemic cells related to the cell cycle

Bone marrow cells of five patients with acute myeloid leukemia were fractionated by means of counterflow centrifugation (elutriation). The different fractions were enriched with cells belonging to subsequent stages of the cell cycle. Cytokinetic evaluation of these cell fractions was performed by [³H]thymidine autoradiography, [³H]thymidine incorporation and DNA/RNA-flow cytometry.Phosphorylation of cytosine arabinoside (ara-C, 1-β-d-arabinofuranosylcytosine) in the different fractions was measured by incubation of the cells for 30 min with 1.07 μM [³H]ara-C. Phosphorylation of ara-C in the whole bone marrow samples ranged from 5.9 to 33.2 pmol/10⁶ cells. In the fractions containing only G₁-phase cells, phosphorylation ranged from 1.2 to 19.5 pmol/10⁶ cells. The phosphorylation seems to increase before DNA synthesis starts. Maximal activities were found in the fractions enriched with cells in late G₁- or S-phase of the cell cycle. In these fractions the ara-C phosphorylating activity was 1.5–8 times higher compared to the fractions with the lowest activity.One may therefore assume that not only S-phase cells are killed by ara-C, but that G₁-phase cells which can phosphorylate ara-C, may also be doomed when they enter S-phase, since the elimination of the intracellular cytosine arabinoside tri-phosphate (ara-CTP) is a relatively slow process. The fraction of G₁-phase cells phosphorylating ara-C, may be an important determinant in the extent of the cell-killing effect of ara-C treatment in the different leukemias.     

Influence of dietary carbohydrates on cholesterol metabolism in casein-fed rabbits

Dietary carbohydrates consisting of cornstarch and dextrose, as well as molassesenriched dextrose, were fed to rabbits in a high fat, cholesterol-free casein purified diet in order to evaluate their influence on food intake, and more specifically on the magnitude of the cholesterol response to casein feeding. Food intake and weight gain were similar in all groups, regardless of carbohydrate origin. The non-significant trend in serum cholesterol elevation in the molasses+dextrose group was however translated by a significant increase in its LDL-cholesterol, compared to the cornstarch and dextrose groups. Moreover, the significant increase in HDL-C and 4,3-fold decrease in the LDL-C/HDL-C ratio induced by cornstarch, in comparison to the dextrose and dextrose+molasses groups, suggests that the atherosclerosis risk associated to casein intake may be attenuated by the use of cornstarch. In addition, significantly higher liver cholesterol and TG levels following molasses+dextrose feeding, compared to dextrose and cornstarch, suggests that molasses enhances the effects of casein on serum cholesterol by a series of reactions originating from an increase in this group's hepatic lipids.     

Three-step isolation of human blood monocytes using discontinuous density gradients of Percoll

A three step method for the purification of normal human blood monocytes is described. The procedure consists of a combination of dextran sedimentation, Ficoll-Isopaque (F-I) centrifugation and isopycnic centrifugation on discontinuous gradients of Percoll. No selective loss of monocytes was observed after the first step, and after F-I centrifugation mononuclear cells (MNC) were obtained, of which 20 +/- 6% were monocytes. The MNC were further separated on hyper-osmotic and iso-osmotic discontinuous density gradients of Percoll. The best purification of monocytes occurred on hyper-osmotic density gradients and the density interface between 1.074 and 1.066 g/ml yielded 85 +/- 7% monocytes, 13 +/- 7% lymphocytes and 1 +/- 1% granulocytes. 77 +/- 16% of the monocytes obtained after F-I centrifugation, were recovered in this interface. The purified monocytes were viable and retained their capacity to mature into macrophages. The whole procedure takes about 5 h, is reproducible and can be applied to small and large volumes (500 ml) of blood.     

黄芩苷发酵转化成黄芩素和千层纸素A的黄芩内生真菌的筛选及鉴定

目的:采用现代微生物发酵转化技术开发传统中药——黄芩,从黄芩植株体内筛选可发酵转化黄芩苷的内生真菌,以期替代传统化学方法获得天然药效成分,同时缓解该中药资源紧张的现状。方法:以新鲜黄芩植株为材料,采用马铃薯葡萄糖琼脂(PDA)平板培养消毒组织块,分离单菌落,黄芩药粉选择性培养法初筛,黄芩苷发酵转化实验复筛,HPLC分析转化产物。结果:从新鲜黄芩植株根部分离得到一株发酵转化黄芩苷的内生真菌R3,该菌在添加0.1%黄芩苷的马铃薯葡萄糖培养液(PDB)中能转化黄芩苷,同时产生黄芩素和千层纸素A共2种产物,于28℃,150 r·min~(-1)培养5 d转化率61.09%,其中黄芩素和千层纸素A的摩尔比3∶5。利用形态分析及显微鉴定并结合分子生物学技术手段,鉴定该菌为青霉菌,定名为Penicillium sp. R3。结论:筛选得到黄芩内生真菌Penicillium sp. R3,该菌能够转化黄芩苷得到活性更强、更易吸收的黄芩素,以及近年来因更多药效不断被发现而备受关注的千层纸素A,具有一定的研究价值与应用价值。     

A multi-center randomized trial of two different intravenous fluids during labor

Objective: To determine if the intrapartum use of a 5% glucose-containing intravenous solution decreases the chance of a cesarean delivery for women presenting in active labor.

Methods: This was a multi-center, prospective, single (patient) blind, randomized study design implemented at four obstetric residency programs in Pennsylvania. Singleton, term, consenting women presenting in active spontaneous labor with a cervical dilation of <6?cm were randomized to lactated Ringer's with or without 5% glucose (LR versus D5LR) as their maintenance intravenous fluid. The primary outcome was the cesarean birth rate. Secondary outcomes included labor characteristics, as well as maternal or neonatal complications.

Results: There were 309 women analyzed. Demographic variables and admitting cervical dilation were similar among study groups. There was no significant difference in the cesarean delivery rate for the D5LR group (23/153 or 15.0%) versus the LR arm (18/156 or 11.5%), [RR (95% CI) of 1.32 (0.75, 2.35), p?=?0.34]. There were no differences in augmentation rates or intrapartum complications.

Conclusions: The use of intravenous fluid containing 5% dextrose does not lower the chance of cesarean delivery for women admitted in active labor.     

Blood transfusion-induced suppression of cellular immunity in man

The effect of planned blood transfusions on cell-mediated immunity was studied in previously nontransfused prospective kidney graft recipients. Following transfusion of washed erythrocytes a marked suppression of cellular immunity was found, indicated by reduced response to mitogenic (PHA, Con A, PWM) and antigenic stimulation (Ag-C containing PPD, tetanus toxoid, streptolysin, mumps, vaccinia antigen). A second transfusion led to a more pronounced and prolonged immunosuppression. No suppression was found when autologous blood was applied to volunteers. Preliminary results show autologous and allogeneic MLR suppression when mitomycin-C treated patient cells taken after transfusion are added. Our findings indicate that blood transfusion-induced suppression of cell-mediated immunity might be caused by an unspecific suppressor cell.