Surfactant-induced breakthrough effects during the operation of two-phase biocatalytic membrane reactors

Surface-active components, both reactants and products, are frequently encountered in two-phase, aqueous-organic, biocatalytic reactions, When such reaction are carried out in a membrane reactor, employing a membrane selectively wetted by one of the two reactants, changes in the content of these surfactants- as a consequence of the progress of the reaction-can lead to wetting transitions at the two membrane-liquid interfaces as a result of adsorption of the tenside. This can lead to a decrease in the pressure required to cause the, initially, nonwetting phase to break through the membrane. Such effects render difficult the operation of two-phase membrane bioreactors. Hence, it is necessary to make a careful selection of the membrane material and type by considering factors such as UF versus MF and low MWCO versus high MWCO to enable the reactor to be operated without breakthrough, but without significantly compromising the reaction rates that can be maintained.The phenomena leading to breakthrough effects are discussed in this paper, and experimental results for the hydrolysis of ethyl laurate by lipase from Candida rugosa in a batch flat sheet membrane reactor are presented with the reactor operated with a variety of membranes. An experimental result showing the decrease in the pressure required to cause breakthrough of the organic phase (for the system ethyl laurate-lauric acid-water) as the content of the highly surface-active lauric acid in the organic phase is increased is also presented for an asymmetric, hydrophilic meta-aramid ultrafiltration membrane. (c) 1994 John Wiley & Sons, Inc.     

A study of the reproducibility of a fluorescence in situ hybridization bladder cancer detection assay

OBJECTIVE: To assess the reproducibility of the UroVysion fluorescence in situ hybridization (FISH) bladder cancer detection assay. STUDY DESIGN: Thirteen specimens (2 negative, 3 low-level positive [1-10% abnormal cells], 5 mid-level positive [11-75%], and 3 high-level positive [>75%]) were analyzed by 7 cytotechnologists. Each cytotechnologist rendered an overall diagnosis of positive or negative and determined the percentage of abnormal urothelial cells for all positive specimens. RESULTS: The interobserver reproducibility of the assay was 100% for mid-level and high-level positive specimens, 93% for negative specimens, and 78% for low-level positive specimens. The range of percent abnormal determinations was highest for mid-level positive specimens, with mean SDs of 1.8%, 16.4% and 10.1% for the low-, mid-, and high-level positives, respectively. CONCLUSION: There was a high level of reproducibility among the mid- and high-level positive specimens. The reproducibility for low-level positive specimens was lowest, suggesting that such specimens should be reviewed by a second technologist to ensure an accurate diagnosis. The findings of this study are important for further elucidating the clinical value of quantitative FISH analysis in the management of patients undergoing FISH testing for bladder cancer.     

人微小病毒3个主要蛋白基因真核表达载体的构建

目的:分别克隆人细小病毒B19三个主要蛋白VP1、VP2、NS1全长基因,构建真核表达载体。方法:利用PCR和分子克隆技术,分别将B19病毒vp1、vp2、ns1基因全长片段扩增后,构建带荧光标签的真核表达载体;在人体细胞中表达并通过荧光、RT-PCR和Western Blot、测序等方法鉴定。结果:成功构建了包含B19病毒vp1、vp2、ns1全长基因,并在人体细胞中表达了VP1、VP2、NS1蛋白。结论:人微小病毒B19三个主要蛋白基因得到克隆和表达,为进行相关的研究奠定了基础。     

Membrane properties of plant sterols in phospholipid bilayers as determined by differential scanning calorimetry, resonance energy transfer and detergent-induced solubilization

The increased use of plant sterols as cholesterol-lowering agents warrants further research on the possible effects of plant sterols in membranes. In this study, the effects of the incorporation of cholesterol, campesterol, beta-sitosterol and stigmasterol in phospholipid bilayers were investigated by differential scanning calorimetry (DSC), resonance energy transfer (RET) between trans parinaric acid (tPA) and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC), and Triton X-100-induced solubilization. The phospholipids used were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), D-erythro-N-palmitoyl-sphingomyelin (PSM), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In DSC experiments, it was demonstrated that the sterols differed in their effect on the melting temperatures of both the sterol-poor and the sterol-rich domains in DPPC and PSM bilayers. The plant sterols gave rise to lower temperatures of both transitions, when compared with cholesterol. The plant sterols also resulted in lower transition temperatures, in comparison with cholesterol, when sterol-containing DPPC and PSM bilayers were investigated by RET. In the detergent solubilization experiments, the total molar ratio between Triton X-100 and POPC at the onset of solubilization (R(t,sat)) was higher for bilayers containing plant sterols, in comparison with membranes containing cholesterol. Taken together, the observations presented in this study indicate that campesterol, beta-sitosterol and stigmasterol interacted less favorably than cholesterol with the phospholipids, leading to measurable differences in their domain properties.     

Preparation of new lipases derivatives with high activity–stability in anhydrous media: adsorption on hydrophobic supports plus hydrophilization with polyethylenimine

A novel method to prepare immobilized lipases derivatives is hereby proposed. Lipases are firstly adsorbed on supports having large internal surfaces covered by hydrophobic groups (e.g. polyacrylic resins covered by C18 moieties). Then, immobilized lipases are incubated in the presence of polyethyleneimine (PEI) at a pH value over the isoelectric point of the enzyme in order to cover the lipase surface with this polymer. In this way, we try to minimize all possible direct interactions between immobilized lipase and organic solvents when using these derivatives in anhydrous media.

Lipases from Rhizomucor miehie (RML) and Candida rugosa (CRL) were immobilized according to the proposed protocol. These derivatives were very active and very stable when catalyzing esterifications and transesterifications in anhydrous media. For example, RML derivatives exhibited a very high synthetic activity (more than 1000 Units/g immobilized biocatalyst) even when catalyzing the esterification of lauric acid with octanol at water activity values very close to zero. On the contrary, covalently immobilized derivatives exhibited a much lower synthetic activity under similar conditions (less than 10 Units/g of immobilized biocatalyst). Moreover, these new RML derivatives preserve 100% activity after incubation for 3 days in anhydrous butanone in the presence of molecular sieves. Under the same conditions, commercial immobilized RML lost more than 90% of activity in less than 10 min.     

Tiludronate treatment improves structural changes and symptoms of osteoarthritis in the canine anterior cruciate ligament model

Introduction

The aim of this prospective, randomized, controlled, double-blind study was to evaluate the effects of tiludronate (TLN), a bisphosphonate, on structural, biochemical and molecular changes and function in an experimental dog model of osteoarthritis (OA).

Methods

Baseline values were established the week preceding surgical transection of the right cranial/anterior cruciate ligament, with eight dogs serving as OA placebo controls and eight others receiving four TLN injections (2 mg/kg subcutaneously) at two-week intervals starting the day of surgery for eight weeks. At baseline, Week 4 and Week 8, the functional outcome was evaluated using kinetic gait analysis, telemetered locomotor actimetry and video-automated behaviour capture. Pain impairment was assessed using a composite numerical rating scale (NRS), a visual analog scale, and electrodermal activity (EDA). At necropsy (Week 8), macroscopic and histomorphological analyses of synovium, cartilage and subchondral bone of the femoral condyles and tibial plateaus were assessed. Immunohistochemistry of cartilage (matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS5)) and subchondral bone (cathepsin K) was performed. Synovial fluid was analyzed for inflammatory (PGE₂ and nitrite/nitrate levels) biomarkers. Statistical analyses (mixed and generalized linear models) were performed with an α-threshold of 0.05.

Results

A better functional outcome was observed in TLN dogs than OA placebo controls. Hence, TLN dogs had lower gait disability (P = 0.04 at Week 8) and NRS score (P = 0.03, group effect), and demonstrated behaviours of painless condition with the video-capture (P < 0.04). Dogs treated with TLN demonstrated a trend toward improved actimetry and less pain according to EDA. Macroscopically, both groups had similar level of morphometric lesions, TLN-treated dogs having less joint effusion (P = 0.01), reduced synovial fluid levels of PGE₂ (P = 0.02), nitrites/nitrates (P = 0.01), lower synovitis score (P < 0.01) and a greater subchondral bone surface (P < 0.01). Immunohistochemical staining revealed lower levels in TLN-treated dogs of MMP-13 (P = 0.02), ADAMTS5 (P = 0.02) in cartilage and cathepsin K (P = 0.02) in subchondral bone.

Conclusion

Tiludronate treatment demonstrated a positive effect on gait disability and joint symptoms. This is likely related to the positive influence of the treatment at improving some OA structural changes and reducing the synthesis of catabolic and inflammatory mediators.     

New solution method for equations of reaction and mass transfer in biocatalyst particles

Summary For numerical solution of the reaction-mass transfer equations for immobilised biocatalysts it may be better to start integration at the particle surface and proceed inwards: calculations are targetted on the region to which practically interesting changes are often confined (because concentrations are effectively zero in the interior); and during iterative solution wrong initial estimates may be rejected after detecting anomalies early in the integration.Symbols C_b substrate concentration in bulk (mol m^–3) - c dimensionless substrate concentration (C/C_b) (-) - D_e effective diffusion coefficient (m²s^–1) - Da Damkohler number (V.r_o ²/D_e.K_s) (-) - K_s substrate concentration kinetic coefficient (mol m^–3) - k_e external mass transfer coefficient (ms^–1) - r_o bead radius (m) - Sh Sherwood number (k_e.r_o/D_e) (-) - V maximum rate per unit volume in beads (mol m^–3s^–1) - x dimensionless distance from bead centre (r/r_o) (-) - dimensionless kinetic coefficient (K_s/C_b) (-) - _o effectiveness factor (-)     

Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.     

The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas

A comprehensive analysis of somatic and germline mutations related to DNA mismatch-repair (MMR) genes can clarify the prevalence and mechanism of inactivation in colorectal carcinoma (CRC). In the present study, 257 unselected patients referred for CRC resection were examined for evidence of defective DNA MMR. In particular, we sought to determine the frequency of hereditary defects in DNA MMR in this cohort of patients. MMR status was assessed by testing of tumors for the presence or absence of hMLH1, hMSH2, and hMSH6 protein expression and for microsatellite instability (MSI). Of the 257 patients, 51 (20%) had evidence of defective MMR, demonstrating high levels of MSI (MSI-H) and an absence of either hMLH1 (n=48) or hMSH2 (n=3). All three patients lacking hMSH2, as well as one patient lacking hMLH1, also demonstrated an absence of hMSH6. DNA sequence analysis of the 51 patients with defective MMR revealed seven germline mutations-four in hMLH1 (two truncating and two missense) and three in hMSH2 (all truncating). A detailed family history was available for 225 of the 257 patients. Of the seven patients with germline mutations, only three had family histories consistent with hereditary nonpolyposis colorectal cancer. Of the remaining patients who had tumors with defective MMR, eight had somatic mutations in hMLH1. In addition, hypermethylation of the hMLH1 gene promoter was present in 37 (88%) of the 42 hMLH1-negative cases available for study and in all MSI-H tumors that showed loss of hMLH1 expression but no detectable hMLH1 mutations. Our results suggest that, although defective DNA MMR occurs in approximately 20% of unselected patients presenting for CRC resection, hereditary CRC due to mutations in the MMR pathway account for only a small proportion of patients. Of the 257 patients, only 5 (1.9%) appear to have unequivocal evidence of hereditary defects in MMR. The epigenetic (nonhereditary) mechanism of hMLH1 promoter hypermethylation appears to be responsible for the majority of the remaining patients whose tumors are characterized by defective DNA MMR.