Methemoglobin Reduction in Crocodile Blood: Are High Levels of MetHb Typical of Healthy Reptiles?

In 82 wild-caught Crocodylus porosus, levels of NADH-MetHb reductase and GSH seem adequate to maintain hemoglobin in its reduced functional state. Studies of C. porosus erythrocytes in vitro show reduction of metHb in the presence of lactate, glucose and plasma, but not pyruvate. These findings, together with recent data which show low metHb in a variety of reptiles, cast doubt on the accepted view that high levels of MetHb are typical of healthy reptiles. One explanation for the sharp contrast between earlier and more recent data could be technical. We found low metHb in Crocodylus johnstoni, Chelodina longicollis and Sphenomorphus quoyi. However, high and variable values reminiscent of many of the earlier data were obtained by omitting final centrifugation prior to spectrophotometry. Interestingly, this step is not part of the standard clinical method but is necessary in analyses of blood with nucleated red cells. These observations suggest that high metHb may not be typical of reptiles after all.

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

A simplified, sequential, phosphorus fractionation method

Hedley er al. (1982) developed what has become the most widely used land modified), phosphorus (P) fractionation technique. It consists of sequential extraction of increasingly less phytoavailable P pools. Extracts are centrifuged at up to 25000 g (RCF) and filtered to 0.45 mu m to ensure that soil is not lost between extractions. In attempting to transfer this method to laboratories with limited facilities, it was considered that access to high-speed centrifuges, and the cost of frequent filtration may prevent adoption of this P fractionation technique. The modified method presented here was developed to simplify methodology, reduce cost, and therefore increase accessibility of P fractionation technology. It provides quantitative recovery of soil between extractions, using low speed centrifugation without filtration. This is achieved by increasing the ionic strength of dilute extracts, through the addition of NaCl, to flocculate clay particles. Addition of NaCl does not change the amount of P extracted. Flocculation with low speed centrifugation produced extracts comparable with those having undergone filtration (0.025 mu m). A malachite green colorimetric method was adopted for inorganic P determination, as this simple manual method provides high sensitivity with negligible interference from other anions. This approach can also be used for total P following digestion, alternatively non-discriminatory methods, such as inductively coupled plasma atomic emission spectroscopy, may be employed.

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: Electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

Lipid microspheres loaded with antigenic membrane proteins of the Leishmania amazonensis as a potential biotechnology application

Lipid microspheres (LM) are excellent drug delivery or vaccines adjuvant systems and are relatively stable. The aim of this work is to develop and characterize a system that is able to encapsulate and present antigenic membrane proteins from Leishmania amazonensis. Membrane proteins are important for vaccine`s formulation because these proteins come in contact with the host cell first, triggering the cell mediated immune response. This is a useful tool to avoid or inactivate the parasite invasion. The LM are constituted by soybean oil (SO), dipalmitoylphosphatidilcholine (DPPC), cholesterol and solubilized protein extract (SPE). The particles formed presented an average diameter of 200 run, low polydispersion and good stability for a period of 30 days, according to dynamic light scattering assays. Isopycnic density gradient centrifugation of LM-protein showed that proteins and lipids floated in the sucrose gradient (5-50%w/v) suggesting that the LM-protein preparation was homogeneous and that the proteins are interacting with the system. The results show that 85% of SPE proteins were encapsulated in the LM. Studies of cellular viability of murine peritoneal macrophages show that our system does not present cytotoxic effect for the macrophages and still stimulates their NO production (which makes its application as a vaccine adjuvant possible). LM-protein loaded with antigenic membrane proteins from L. amazonensis seems to be a promising vaccine system for immunization against leishmaniasis. (C) 2009 Elsevier Inc. All rights reserved.

Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Binding and internalization of the melanocyte stimulating hormone receptor ligand [Nle(4), D-Phe(7)]alpha-MSH in B16 melanoma cells

The current study aims to ascertain the fate of the melanocyte stimulating hormone (MSH) receptor and its ligand [Nle(4), D-Phe(7)]alpha-MsH (NDP-MSH) following binding to murine B16 melanoma cells. Cells were incubated with [I-125]-NDP-MSH for up to 180 min and binding, internalization and degradation determined. Intracellular trafficking of the radiolabel was assessed !using Percoll density gradient centrifugation of homogenized cells. Receptor down-regulation and receptor mRNA levels were also measured over 96 hr after exposure to 1 mu M ligand. NDP-MSH accumulation increased with time in a temperature-dependent manner and was inhibited by excess peptide. The ligand was rapidly internalized and translocated to the lysosomal compartment where it was degraded. Internalization was accompanied by a loss or down-regulation of cell surface receptors, suggesting internalization of the NDP-MSH-receptor complex. No recycling of the receptors between the plasma membrane and intracellular compartments could be detected in this cell-hue. Approximately 15% of the surface receptors were resistant to down-regulation, possibly indicating receptor heterogeneity. Down-regulation persisted ibr up to 96 hr and was accompanied by a decrease in MSH receptor mRNA levels 48 hr after treatment. However, before this time, transcript levels were the same in treated and control cells. In contrast to what was seen with NDP-MSH, cell surface receptors removed with trypsin wc:re rapidly replaced. These results show that NDP-MSH not only induced MSH receptor :internalization but also inhibited receptor turnover, resulting in a prolonged down-regulation. It is concluded that, in B16 cells, the MSH receptor undergoes ligand-dependent internalization, resulting in a prolonged down-regulation. Copyright (C) 1996 Elsevier Science Ltd

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photosedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/Sigma) from 1.32 x 10(-9) m s(-1) to 3.97 x 10(-9) m s(-1) leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s(-1), increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.26 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

Cross-linking studies and membrane localization and assembly of radiolabelled large mechanosensitive ion channel (MscL) of Escherichia coli

The gene encoding the large conductance mechanosensitive ion channel (MscL) of Escherichia coli and several deletion mutants of mscL were cloned under the control of the T7 RNA polymerase promoter. Transformation of these constructs into an E. coli strain carrying an inducible T7 RNA polymerase gene allowed the specific production and labelling of MscL with [S-35]methionine. Preparation of membrane fractions of E. coli cells by sucrose gradient centrifugation indicated that the radiolabelled MscL was present in the inner cytoplasmic membrane in agreement with results of several studies. However, treatment of the labelled cells and cell membrane vesicles with various cross-linkers resulted in the majority of labelled protein migrating as a monomer with a small proportion of molecules (approximate to 25%) migrating as dimers and higher order multimers. This result is in contrast with a finding of a study suggesting that the channel exclusively forms hexamers in the cell membrane off. coli (1) and therefore may have profound implication for the activation and/or ''multimerization'' of the channel by mechanical stress exerted to the membrane. In addition, from the specific activity of the radiolabelled protein and the amount of protein in the cytoplasmic membrane fraction we estimated the number of MscL ion channels expressed under these conditions to be approximately 50 channels per single bacterium. (C) 1997 Academic Press.

Early Increase in Myocardial Perfusion After Stem Cell Therapy in Patients Undergoing Incomplete Coronary Artery Bypass Surgery

Incomplete revascularization is associated with worse long-term outcomes. Autologous bone marrow cells (BMC) have recently been tested in patients with severe coronary artery disease. We tested the hypothesis that intramyocardial injection of autologous BMC increases myocardial perfusion in patients undergoing incomplete coronary artery bypass grafting (CABG). Twenty-one patients (19 men), 59 +/- 7 years old, with limiting angina and multivessel coronary artery disease (CAD), not amenable to complete CABG were enrolled. BMC were obtained prior to surgery, and the lymphomonocytic fraction separated by density gradient centrifugation. During surgery, 5 mL containing 2.1 +/- 1.3 x 10(8) BMC (CD34+ = 0.8 +/- 0.3%) were injected in the ischemic non-revascularized myocardium. Myocardial perfusion was assessed by magnetic resonance imaging (MRI) at baseline and 1 month after surgery. The increase in myocardial perfusion was compared between patients with < 50% (group A, n = 11) with that of patients with > 50% (group B, n = 10) of target vessels (stenosis a parts per thousand yenaEuro parts per thousand 70%) successfully bypassed. Injected myocardial segments included the inferior (n = 12), anterior (n = 7), and lateral (n = 2) walls. The number of treated vessels (2.3 +/- 0.8) was significantly smaller than the number of target vessels (4.2 +/- 1.0; P < 0.0001). One month after surgery, cardiac MRI showed a similar reduction (%) in the ischemic score of patients in group A (72.5 +/- 3.2), compared to patients in group B (78.1 +/- 3.2; P = .80). Intramyocardial injection of autologous BMC may help increase myocardial perfusion in patients undergoing incomplete CABG, even in those with fewer target vessels successfully treated. This strategy may be an adjunctive therapy for patients suffering from a more advanced (diffuse) CAD not amenable for complete direct revascularization.

A mathematical model for Escherichia coli debris size reduction during high pressure homogenisation based on grinding theory

Experimental data for E. coli debris size reduction during high-pressure homogenisation at 55 MPa are presented. A mathematical model based on grinding theory is developed to describe the data. The model is based on first-order breakage and compensation conditions. It does not require any assumption of a specified distribution for debris size and can be used given information on the initial size distribution of whole cells and the disruption efficiency during homogenisation. The number of homogeniser passes is incorporated into the model and used to describe the size reduction of non-induced stationary and induced E. coil cells during homogenisation. Regressing the results to the model equations gave an excellent fit to experimental data ( > 98.7% of variance explained for both fermentations), confirming the model's potential for predicting size reduction during high-pressure homogenisation. This study provides a means to optimise both homogenisation and disc-stack centrifugation conditions for recombinant product recovery. (C) 1997 Elsevier Science Ltd.

Effects of semen storage and separation techniques on sperm DNA fragmentation

Objective: To determine the effect of semen storage and separation techniques on sperm DNA fragmentation. Design: Controlled clinical study. Setting: An assisted reproductive technology laboratory. Patient(s): Thirty normoozospermic semen samples obtained from patients undergoing infertility evaluation. Intervention(s): One aliquot from each sample was immediately prepared (control) for the sperm chromatin dispersion assay (SCD). Aliquots used to assess storage techniques were treated in the following ways: snap frozen by liquid nitrogen immersion, slow frozen with Tris-yolk buffer and glycerol, kept on ice for 24 hours or maintained at room temperature for 4 and 24 hours. Aliquots used to assess separation techniques were processed by the following methods: washed and centrifuged in media, swim-up from washed sperm pellet, density gradient separation, density gradient followed by swim-up. DNA integrity was then measured by SCD. Main Outcome Measure(s): DNA fragmentation as measured by SCD. Result(s): There was no significant difference in fragmentation among the snap frozen, slow frozen, and wet-ice groups. Compared to other storage methods short-term storage at room temperature did not impact DNA fragmentation yet 24 hours storage significantly increased fragmentation. Swim-up, density gradient and density gradient/swim-up had significantly reduced DNA fragmentation levels compared with washed semen. Postincubation, density gradient/swim-up showed the lowest fragmentation levels. Conclusion(s): The effect of sperm processing methods on DNA fragmentation should be considered when selecting storage or separation techniques for clinical use. (Fertil Steril (R) 2010;94:2626-30. (C) 2010 by American Society for Reproductive Medicine.)

Adipose tissue mesenchymal stem cell expansion in animal serum-free medium supplemented with autologous human platelet lysate

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered for human regenerative therapy applications, and safe culture and expansion protocols are needed especially in the context of interspecies contamination. Human platelet lysate (PL) has been proposed as animal serum substitute during in vitro MSC expansion. In this work, a simplified and efficient method to obtain autologous PL to replace animal serum in cell culture applications is described. STUDY DESIGN AND METHODS: PL obtained by freezing and centrifugation procedures was tested as medium supplement for human adipose mesenchymal stem cell (hASC) culture. Differential proliferation, immunophenotypic changes, and differentiation under PL or fetal bovine serum (FBS) were assessed. RESULTS: In contrast to 10% FBS supplementation, cell population doubling time was significantly lower when hASCs were cultured with the same concentration of PL ( PL 22.9 +/- 1.5 hr vs. FBS 106.7 +/- 6.5 hr, t test, p < 0.05). Furthermore, hASCs maintained with 2.5% PL supplementation also showed satisfactory results. Immunophenotypic analysis revealed no differences between hASCs cultivated with PL or FBS supplementation and both cultures retained the potential to differentiate into adipose cells. These results demonstrate that autologous PL obtained from the same donor can be used as animal serum substitute in hASC culture. CONCLUSIONS: Taken together, evidence is provided that platelets provided by a single donor are sufficient to obtain PL for hASC propagation for clinical-scale applications mitigating the potential untoward side effects associated with the use of animal-derived reagents.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.