Density Gradient Centrifugation Technique Research Articles

Leukocytospermia and sperm preparation - a flow cytometric study

BackgroundLeukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.MethodsSemen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm.ResultsBoth sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters) after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical microscopy was satisfactory, the sensitivity of peroxidase test for the detection of leukocytospermia resulted low.ConclusionSeminal leukocytes do not seem to influence sperm preparation results. However, for assisted conception, semen samples containing leukocytes should be processed using swim-up method. Although peroxidase-test is recommended by WHO as the standard method for determining semen leukocytes, it should not be used in clinical research study.

부유밀도구배 원심분리를 이용하여 말초혈액단핵구로부터 고순도 및 고수율의 단세포 분리방법

본 연구에서는, PBMC로부터 고순도 및 고수율의 단세포를 분리하기 위해 Histopaque 용액의 density 및 osmolarity를 PBS와 NaCI 용액으로 1.072 g/ml 및 335 mOsm로 조절하여 간단하고 경제적인 방법인 부유밀도구배 원심분리법을 개발하였다. 위와 같이 조절된 Histopaue 용액을 사용하여 부유밀도구배 원심분리를 시행한 결과 단세포는 Top층에서 가장 많이 회수되었다. 회수된 단세포의 순도는 71.44<TEX>${\sim}$</TEX>82.38%의 범위를 나타내었고 평균 74.75<TEX>${\pm}$</TEX>3.84% 이었다. 또한, 수율은 21.02<TEX>${\sim}$</TEX>53.63%의 범위를 나타내어 평균 32.62<TEX>${\pm}$</TEX>11.16% 이었다. 이러한 방법의 부유밀도구배 원심분리에 의해 분리된 단세포를 수지상세포로 분화 유도시켜 그 형태, 표현형, 기능적인 면을 분석해 보았을 때 전형적인 수지상세포의 기능을 나타내었다. 결론적으로, PBMC로부터 단세포를 분리하기 위해 본 연구에서 사용된 부유밀도구배 원심분리 방법은 plastic adherence, elutriation 및 immunomagnetic selection보다 용이하고 경제적인 방법이다. 나아가 이러한 장점을 바탕으로 임상단계에서 수지상세포를 안전하고, 효율적으로 배양할 수 있는 closed system에도 적용이 가능할 것으로 기대된다. In this work, a simple, inexpensive and reproducible technique of flotation density gradient centrifugation was developed to isolate monocytes with high purity and yield from peripheral blood mononuclear cells (PBMC) using Histopaque solution, density and osmolarity of which were modified to 1.072 g/ml and 335 mOsm with phosphate-buffered saline (PBS) and sodium chloride (NaCl) solution, respectively. The average purity of monocytes was 74.75<TEX>${\pm}$</TEX>3.84%, with the individual purity ranging from 71.44% to 82.38%. The average yield of monocytes was 32.62<TEX>${\pm}$</TEX>11.16%, with the individual yields ranging from 21.02 to 53.63%. The monocytes isolated by floatation density gradient centrifugation could be successfully cultured into morphologically, phenotypically and functionally dendritic cells in vitro. In conclusion, the entire procedure seemed to be faster and more convenient, simple and cost-effective than other monocyte isolation methods, including plastic adherence and density gradient methods, and has the potential to be developed as a closed system for clinical scale generation of dendritic cells.

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.

Percoll density gradient centrifugation and consecutive flow cytometry do not identify leukocytes and leukocyte subtypes in ejaculate specimens.

This paper describes an attempt to establish a new combined method of leukocyte analysis in human ejaculate by Percoll density gradient centrifugation and consecutive flow cytometry. As a first step, leukocyte separation was performed by Percoll density gradient centrifugation with consecutive enrichment of leukocytes, especially granulocytes, in the 40%/60% and 60%/80% Percoll interfaces. Then these fractions were stained with specific monoclonal antibodies and analysed in a Facscan flow cytometer. Flow cytometric analysis did not demonstrate identifiable leukocyte populations, indicating a questionable cross-reaction with spermatozoal elements. Therefore, the combined technique of Percoll density gradient centrifugation and flow cytometric analysis were considered unsuitable for clinical leukocyte determination.

Intrauterine insemination with semen of oligozoospermic men: effectiveness of the continuous-step density gradient centrifugation technique

There has been an argument as to whether the intrauterine insemination (IUI) substantially improves pregnancy rate in cases of oligoasthenozoospermia. The present study evaluates the efficiency of the IUI with the use of the life table analysis. The insemination was performed after washing and concentrating sperm using the continuous-step density gradient centrifugation reported in our previous papers. The number of IUI cycles studied was 222 (46 couples), in 13 cases of which pregnancy was established with a pregnancy rate of 28.3%. The cumulative probability of conception reached to 57% at the eleventh cycles of the IUI treatment. These results suggest that the potential efficiency of the IUI for the oligo-asthenozoospermic cases is high if the sperm is processed in a proper method.

Désérythrocytation de moelle osseuse par gradient de densité sur COBE 2991 avec le kit triple « bérets »

ABO-incompatible bone marrow transplantation requires red blood cell depletion. Lots of laboratory adopted the technique of density gradient centrifugation (Ficoll-hypaque) using the COBE 2991 cell processor with simple-bag processing set. However, tubing of this set is not adapted to the currently available peristaltic pumps. Moreover, two other sets are required: one for the buffy-coat and one for postgradient cell washing. We developed a method using triple-bag processing set to conduct whole-step procedure (concentration, Ficoll and washing). Peristaltic PVC tubing is provided in one line of the set allowing a safe processing without several connections thus reducing risks of microbial contamination. First, we used buffy-coat of total blood for training, then, we carried out red cell depletion of healthy bone marrow donors. The red blood cell depletion was 97.9 ± 1.1% and CD34+ recovery was 89.6 ± 8.7%. These results are very close to those obtained with the simple-bag set (red cell depletion. = 94.0 ± 6.8% and CD34+ recovery = 95.9 ± 20.3%). We conclude that the triple-bag system, very little used in France, is practical, simplified the manipulation and is more safety than the simple-bag set.

Isolation and characterization of dendritic cells from adenoids of children with otitis media with effusion

Dendritic cells were enriched from adenoids of children with otitis media with effusion (OME) by density gradient centrifugation and culture techniques. An enrichment of 40-140-fold was obtained for dendritic cells. These cells were identified using morphology, enzyme cytochemistry, immunocytochemistry and functional criteria. Dendritic cells could be easily distinguished from macrophages. It appeared that the MoAb EBM11 (CD68) discriminated between dendritic cells and macrophages; in dendritic cells this activity was localized in a spot, whereas in macrophages it was found throughout the whole cytoplasm. The fractions enriched with dendritic cells showed a strong stimulatory effect on allogeneic T cells. These responses were MHC class II dependent since they could be blocked by anti-HLA-DR/DQ MoAbs. The data clearly show that dendritic cells from adenoids of children with OME still have functional capacities.

Cell quality in the ASTAMI study

Schachinger et al. 1 recently reported clinical improvement after treatment of acute myocardial infarction with intracoronary injection of autologous bone-marrow-derived mononuclear cells (MNCs) (the REPAIR-AMI Trial). Here, the authors discuss whether lack of improvement in global left ventricular function in our study, the ASTAMI study,2 could be related to impaired cell quality or insufficient cell numbers. We believe this is unlikely for the following reasons: 1. Comparable density gradient centrifugation techniques …

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.

Mild episodes of tourniquet-induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers

BackgroundMonocytes and neutrophils are examples of phagocytic leukocytes, with neutrophils being considered as the 'chief' phagocytic leukocyte. Both monocytes and neutrophils have been implicated to play a key role in the development of ischaemia-reperfusion injury, where they are intrinsically involved in leukocyte-endothelial cell interactions. In this pilot study we hypothesised that mild episodes of tourniquet induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers.MethodsTen healthy human volunteers were recruited after informed consent. None had any history of cardiovascular disease with each subject volunteer participating in the study for a 24 hour period. Six venous blood samples were collected from each subject volunteer at baseline, 10 minutes ischaemia, 5, 15, 30, 60 minutes and 24 hours reperfusion, by means of a cannula from the ante-cubital fossa. Monocyte and neutrophil leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte trapping was investigated by measuring the concentration of leukocytes in venous blood leaving the arm. The cell surface expression of CD62L (L-selectin), CD11b and the intracellular production of hydrogen peroxide (H2O2) were measured via flow cytometry. C-reactive protein (CRP) was measured using a clinical chemistry analyser. Plasma concentrations of D-dimer and von Willebrand factor (vWF) were measured using enzyme-linked fluorescent assays (ELFA).ResultsDuring ischaemia-reperfusion injury, there was a decrease in CD62L and an increase in CD11b cell surface expression for both monocytes and neutrophils, with changes in the measured parameters reaching statistical significance (p =< 0.05). A significant decrease in peripheral blood leukocyte concentration was observed during this process, which was measured to assess the degree of leukocyte trapping in the micro-circulation (p =< 0.001). There was an increase in the intracellular production of H2O2 production by leukocyte sub-populations, which was measured as a marker of leukocyte activation. Intracellular production of H2O2 in monocytes during ischaemia-reperfusion injury reached statistical significance (p = 0.014), although similar trends were observed with neutrophils these did not reach statistical significance. CRP was measured to assess the inflammatory response following mild episodes of ischaemia-reperfusion injury and resulted in a significant increase in the CRP concentration (p =< 0.001). There were also increased plasma concentrations of D-dimer and a trend towards elevated vWF levels, which were measured as markers of coagulation activation and endothelial damage respectively. Although significant changes in D-dimer concentrations were observed during ischaemia-reperfusion injury (p = 0.007), measurement of the vWF did not reach statistical significance.ConclusionTourniquet induced forearm ischaemia-reperfusion injury results in increased adhesiveness, trapping and activation of leukocytes. We report that, even following a mild ischaemic insult, this leukocyte response is immediately followed by evidence of increased inflammatory response, coagulation activity and endothelial damage. These results may have important implications and this pilot study may lead to a series of trials that shed light on the mechanisms of ischaemia-reperfusion injury, including potential points of therapeutic intervention for pathophysiological conditions.

Adenylyl cyclase 5/6 colocalizes with TRPC4 and cell adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell-cell junctions between adjacent pulmonary capillary endothelial cells contribute to the integrity of this barrier and are dynamically regulated by transitions in cAMP. Within PMVECs calcium sensitive adenylyl cyclase activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of adenylyl cyclase and other signaling molecules involved in barrier regulation within the PMVEC membrane has not been described. Here we utilize a detergent free discontinuous sucrose density gradient centrifugation technique to separate caveolae/lipid raft fractions from the bulk plasma membrane. AC5/6 immunoreactivity was detected in the caveolin rich light-density fractions while beta adaptin immunoreactivity was detected in the high-density fraction of non-caveolae regions within the plasma membrane. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in the caveolin rich fraction. In addition, cell adhesion molecules N-cadherin and ALCAM and the scaffolding molecule Dlg localize to caveolae. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and HL60024

Role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury

During this investigation, a model of tourniquet-induced forearm ischaemia-reperfusion injury is employed to investigate the role of leucocytes in damage to the vascular endothelium during ischaemia-reperfusion injury. Leucocyte entrapment is investigated by measuring the concentration of leucocytes in venous blood leaving the arm. Neutrophil and monocyte leucocyte subpopulations are isolated by density gradient centrifugation techniques. Cell surface expression of CD11b and the intracellular production of hydrogen peroxide are measured via flow cytometry. Plasma concentrations of elastase and von Willebrand factor (vWF) are measured using enzyme-linked immunosorbemt assay (ELISA) techniques. During ischaemia-reperfusion, there was an increase in CD11b cell surface expression on neutrophils (P=0.040) and monocytes (P=0.049), and a decrease in peripheral blood leucocytes (P=0.019). There was an increase in the intracellular production of hydrogen peroxide by leucocyte subpopulations (P=0.027 [neutrophils], P=0.091 [monocytes]) and in the plasma elastase concentration (P=0.05). There was also a trend to increasing plasma concentration of vWF (P=0.0562), which was measured as a marker of endothelial damage. Ischaemia-reperfusion results in increased adhesiveness, entrapment and activation of leucocytes. Even following a mild ischaemic insult, this leucocyte response was followed immediately by evidence of endothelial damage. These results may have important implications for understanding the development of chronic diseases that involve mild ischaemic episodes.

A practical method for enumerating cysts of ciliates in natural marine sediments

In order to determine the abundance of intact cysts of planktonic ciliates in natural sediments, we developed a new practical method using autofluorescence of the fixative glutar- aldehyde. After treatment with glutaraldehyde, the cyst walls fluoresced yellowish-green under blue excitation. The fluorescence was always clear in the intact cysts, while only part of the empty cysts was weakly fluorescent. This fluorescence made detection of the intact cysts easy among detrital materials, even for the cysts covered with detritus. It was also easy to distinguish the ciliate cysts morphologically from other organisms that also fluoresced in the treated sediments. This method was compared with the density gradient centrifugation technique, and the advantage of the former in frequent and routine investigations is discussed.

Nurture vs nature: how can we optimize sperm quality?

Nurture vs nature: how can we optimize sperm quality?

Translesion replication in cisplatin-treated xeroderma pigmentosum variant cells is also caffeine-sensitive: features of the error-prone DNA polymerase(s) involved in UV-mutagenesis

Patients with xeroderma pigmentosum variant (XP-V) have a higher risk to skin cancer and XP-V cells are extremely mutable by ultraviolet (UV). The defective gene encodes a DNA polymerase (Polη) which catalyzed relatively accurate translesion synthesis past the cyclobutane dimer of UV-lesions instead of the replicative polymerase(s) that stalled just before the lesion. Pulse-chase studies have shown that translesion replication in XP-V cells is delayed, but does not completely cease. Taking these results together, error-prone polymerase(s) are plausively involved in the UV-mutagenesis in XP-V devoid of Polη. However, less is known about the polymerase(s) in vivo. Using an alkaline sucrose density gradient centrifugation (ASDG) technique, translesion replication is detected in the two XP-V strains XP30RO and XP115LO. As reported by Lehmann et al. [Proc. Natl. Acad. Sci. U.S.A. 72 (1975): 219] in XP-V; (i) smaller replication products were accumulated after UV irradiation; (ii) the elongation of these products was delayed; (iii) the elongation was markedly inhibited by caffeine. XP-V cells UV-irradiated at mid-S phase were normally S-arrested, and no “override” by caffeine (i.e. abrogation of the S-checkpoint) was observed by flow cytometry, suggesting that caffeine does not act via cdc kinase here; (iv) butylphenyldeoxyguanosine (BuPGdR) inhibited elongation of replication products only in UV-irradiated XP-V cells; (v) dideoxycytidine or dideoxyinosine had no effect on this process in either normal or XP-V cells. Next, similar phenomena to UV (all of above i to v) were observed also in cisplatin-treated XP-V cells. Polη was indicated to participate in cisplatin-induced translesion replication in normal cells. Summing up the above results, the polymerase(s) which work in translesion replication in XP-V are probably BuPGdR-sensitive, insensitive to dideoxynucleotides and can bypass also cisplatin-lesions. To date, several polymerases capable of lesion-bypass synthesis have been isolated. The features presented here are quite useful for identifying the error-prone polymerase(s) involved in UV-mutagenesis.

Evidence for a carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles.

Recent studies from our laboratory have demonstrated the presence of a pH-dependent, amiloride-sensitive, electroneutral carrier-mediated exchange for thiamine absorption in the human small intestinal brush-border membrane vesicles. However, the mechanism of thiamine transport across the human small intestinal basolateral membrane is not understood. The present study was aimed to characterize the mechanism of thiamine transport across the basolateral membranes of the human jejunum. Basolateral membrane vesicles (BLMV) were purified from mucosal scrapings of organ donors, utilizing a Percoll continuous density gradient centrifugation technique. The results showed [3H] thiamine uptake into BLMV to be: (1) markedly stimulated in the presence of an outwardly directed H+ gradient (pH 5.5in/7.5out); (2) significantly inhibited by amiloride in a dose-dependent manner; (3) sensitive to temperature and medium osmolarity and insensitive to changes in membrane potential; (4) not influenced by the addition of 1 mM Mg(2+)-ATP, inside and outside the vesicles in the presence of Na+ and K+; (5) inhibited by structural analogs-amprolium, oxythiamin, and unlabeled thiamine (100 microM); (6) not affected by organic cations, eg, TEA, N-methyl-nicotinamide (NMN), and choline; and (7) saturable as a function of concentration (apparent Km of 0.76 +/- 0.21 microM and a V(max) of 1.38 +/- 0.35 pmol/mg protein/10 sec). These results indicate the presence of a proton gradient-dependent specialized carrier-mediated exchange mechanism for thiamine transport across the human jejunum basolateral membranes.

Occurrence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae) in Brazil.

The objective of the present study was to investigate the prevalence of Cryptosporidium (Apicomplexa, Cryptosporidiidae) in the snake Crotalus durissus terrificus (Serpentes, Viperidae). Fifty animals were evaluated for the presence of oocysts of Cryptosporidium sp. at the time of arrival and 30 and 60 days later. Intestinal washings with saline solution (1% body weight), fecal samples, and organ scrapings were collected during the study. Oocysts were concentrated by an ether-phosphate-buffered saline sedimentation technique and then separated by a density gradient centrifugation technique. Smears were made with the sediment and submitted to modified acid-fast and auramine-rhodamine staining. Cryptosporidium-positive smears were used as controls for the experimental findings. The overall prevalence of Cryptosporidium sp. oocysts was 14%. Among the positive snakes, oocysts were detected only in the intestinal washing in two specimens, only in the feces in four specimens, and in both materials at least once in one specimen. The positive snakes were predominantly from Santa Maria da Serra city State of São Paulo (57.1%). We also observed that all of the examinations that presented positive results were obtained at least 27 days after the capture of the animals.

Separation of distinct compartments of rice Golgi complex by sucrose density gradient centrifugation

We studied the separation of distinct compartments of the Golgi complex in rice and tobacco employing the discontinuous sucrose density gradient centrifugation technique with EDTA or MgCl 2. In the presence of EDTA, rice Golgi marker enzymes, nucleoside diphosphatase and N-acetylglucosaminyltransferase were fractionated in the 28 and 33% sucrose fractions, respectively. Peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 that are shown to be located at Golgi complex were broadly distributed around a 31.5% sucrose density. Under a MgCl 2-supplemented condition, the peak of nucleoside diphosphatase was shifted to 29 and 37% sucrose and N-acetylglucosaminyltransferase was distributed in the 37% sucrose fraction. The distribution of peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 were also shifted to the 34.5% sucrose fraction. Furthermore, glucan synthase-I and UDP-glucose pyrophosphorylase were fractionated in the 26.5% sucrose fraction, regardless of the presence or absence of MgCl 2. These results indicate that there exist at least four distinct compartments of the rice Golgi complex. The tobacco Golgi marker enzymes and Golgi-localized proteins were distributed in the 30–31% sucrose fractions in the presence of EDTA and slightly shifted to the 32–33% sucrose fractions under a MgCl 2-supplemented condition. These results suggest that the sedimentation behavior of compartments of the rice Golgi complex in the gradients is specific for rice cells.

Modeling the Classic Meselson &amp; Stahl Experiment

Research Article| May 01 2001 Modeling the Classic Meselson & Stahl Experiment JoBeth D'Agostino JoBeth D'Agostino Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (2001) 63 (5): 358–361. https://doi.org/10.2307/4451124 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation JoBeth D'Agostino; Modeling the Classic Meselson & Stahl Experiment. The American Biology Teacher 1 May 2001; 63 (5): 358–361. doi: https://doi.org/10.2307/4451124 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.

Characterization of differing forms of unburned carbon present in fly ash separated by density gradient centrifugation

It is generally known that the ASTM LOI specification is not sufficient to identify the suitability of a fly ash for the cement industry, since this criterion only gives an approximation to the carbon content of a sample and in many instances provides a poor correlation with the capacity to adsorb air-entrainment admixtures (AEAs). Petrographic examinations have shown that the unburned carbon is not visually uniform and three microscopically distinct carbon types have been identified: inertinite, isotropic coke and anisotropic coke. Concentrates of these three carbon types have been generated from two high-carbon-content fly ash samples using the technique of density gradient centrifugation (DGC). The work reported here represents the first characterization of the bulk properties of these three carbon types, where the highest DGC purity fractions were extensively analyzed by several analytical techniques, including elemental and surface area analyses. The density of like carbon forms isolated from the two fly ashes appears to be quite similar, regardless of the source of the fly ash. The H/C atomic ratios are ∼0.03–0.02, indicating a high degree of condensation. The surface areas are relatively low (10–60 m 2 g −1), and most of the pores are in the mesopore range (2–50 nm in width). For both series of fly ash samples, the surface area was found to increase linearly with increasing particle density. On the basis of the distinctive physical and chemical properties (density, nitrogen and oxygen concentrations, as well as surface area and mesopore volume) of the three carbon types, it is anticipated that each carbon type will have specific adsorption behavior of AEAs.