Bovine Serum Albumin In Phosphate-buffered Saline Research Articles

Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+

In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.

A Synthetic Peptide Ligand of Neural Cell Adhesion Molecule (NCAM), C3d, Promotes Neuritogenesis and Synaptogenesis and Modulates Presynaptic Function in Primary Cultures of Rat Hippocampal Neurons

The neural cell adhesion molecule (NCAM) plays a key role in morphogenesis of the nervous system and in remodeling of neuronal connections accompanying regenerative and cognitive processes. Recently, a new synthetic ligand of NCAM, the C3-peptide, which binds to the NCAM IgI module, has been identified by means of combinatorial chemistry (Rønn, L. C. B, Olsen, M., Ostergaard, S., Kiselyov, V., Berezin, V., Mortensen, M. T., Lerche, M. H., Jensen, P. H., Soroka, V., Saffell, J. L., Doherty, P., Poulsen, F. M., Bock, E., Holm, A., and Saffells, J. L. (1999) Nat. Biotechnol. 17, 1000-1005). In vitro, the dendrimeric form of C3, termed C3d, disrupts NCAM-mediated cell adhesion, induces neurite outgrowth, and triggers intracellular signaling cascades similar to those activated by homophilic NCAM binding. The peptide may therefore be expected to regulate regeneration and synaptic plasticity. Here we demonstrate that in primary cultures of hippocampal neurons: 1) C3d induces a sustained neuritogenic response, the neuritogenic activity of the compound being dependent on the dose, starting time, and duration of peptide application; 2) the peptide triggers the neuritogenic response by forming an adhesive substratum necessary for NCAM-mediated neurite formation and elongation; 3) C3d promotes synapse formation; and 4) C3d modulates the presynaptic function, causing a transient increase of the function at low (2 and 5 microm) doses and a reduction when applied at a higher concentration (10 microm). The effect of the peptide is dependent on the activation of the fibroblast growth factor receptor. We suggest that C3d may constitute a useful lead for the development of compounds for treatment of various neurodegenerative disorders.

Relation of cyclooxygenase isoenzyme expression and coronary artery remodeling

Inflammation plays a key role in coronary atherosclerosis. Increased markers of inflammation have been consistently associated with a worse outcome in patients with clinical or subclinical coronary artery disease. 1 Cyclooxygenase (COX) or prostaglandin synthase G/H are key enzymes responsible for the metabolism of arachidonic acid, and they exist in 2 isoforms—COX-1 and COX-2. COX activity has been described in both healthy human coronary arteries and in association with atherosclerosis and transplant vasculopathy. 2,3 Coronary arteries undergo changes in lumen size in response to coronary atherosclerosis; a phenomenon described as coronary remodeling. 4,5 Coronary remodeling encompasses outward expansion or positive remodeling, and conversely, narrowing or negative remodeling. Positive remodeling, although serving to maintain luminal size and thus possibly maintaining adequate coronary flow, is associated with a higher incidence of unstable coronary syndromes. 6 Although a previous studies 7 has suggested a relation between inflammation and coronary remodeling, the role of the COX pathway has not been explored. We sought to determine the relation between the localization of COX isoenzymes and coronary artery remodeling in patients who underwent directional coronary atherectomy (DCA). ••• The study population consisted of 21 patients who had DCA performed for a single epicardial lesion in a native coronary artery. Clinical presentation was defined as stable or unstable using standard clinical criteria. 8 Intravascular ultrasound imaging was performed using standard techniques as previously reported. 9 All images were obtained using a 30-MHz 3.5Fr monorail ultrasound catheter (Boston Scientific, Watertown, Massachusetts) interfaced with a scanner (Hewlett-Packard, Andover, Massachusetts). After coronary angiography, intravenous heparin and intracoronary nitroglycerin were administered, and the ultrasound catheter was inserted over a guide wire distal to the target lesion site. The ultrasound catheter was then withdrawn manually during continuous imaging, and images were recorded on 1/2-in SuperVHS videotape. The lesion site was identified using cine angiography and verbal annotation. Each piece of tissue was fixed for 8 hours in Histochoice (Amresco, Solon, Ohio). The tissue was then processed and paraffin was embedded according to standard laboratory procedures. Sections were cut from each block at 4 m, collected onto electrostatically charged slides, and baked at 60°C for 60 minutes. Two sections were collected and immunostained for each antibody. The antibodies used in this study were anti-HAM56 (Dako, Carpinteria California), anti-COX-1 (Cayman Chemical, Ann Arbor, Michigan), anti-COX-2 (Cayman Chemical), and anti--actin (Dako). The paraffin was removed from the baked tissue sections in 3 changes of xylene, and the tissue was hydrated through graded alcohols before rinsing them in phosphate-buffered saline (PBS). Immunohistochemical staining was performed using a Jung Histostainer (Leica, Illinois), with processing occurring at 30°C. The first stage of the procedure involved the application of a 1% hydrogen peroxide solution in methanol for 5 minutes to remove any endogenous peroxidase present in the tissue section. A blocking solution comprising a 1:10 dilution of normal rabbit serum (Dako) in PBS was added for 10 minutes before application of the primary antibody. The required dilutions of antibody were prepared using 1% bovine serum albumin in PBS. Incubation occurred at 30°C for 60 minutes, and a 1:200 dilution of biotinylated rabbit anti-mouse polyclonal antibody (Dako) was added for an additional 30 minutes. The antibodies were labeled using an Elite avidin/biotin/peroxidase complex (Vector Laboratories, Burlingame, California) applied for 30 minutes. The final stage comprised the addition of 3,3diaminobenzidine as a chromogen (DAB Kit, Vector

Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse development.

The caspase family of proteases represents the main machinery by which apoptosis occurs. In vitro studies have revealed that upstream caspases are activated in response to apoptotic stimuli, and the active caspases in turn process downstream effector caspases that are involved in the destruction of cellular structure. Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g. during development. We have identified vimentin as the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, showed that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. Our results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death.

Specific Amelogenin Gene Splice Products Have Signaling Effects on Cells in Culture and in Implants in Vivo

Low molecular mass amelogenin-related polypeptides extracted from mineralized dentin have the ability to affect the differentiation pathway of embryonic muscle fibroblasts in culture and lead to the formation of mineralized matrix in in vivo implants. The objective of the present study was to determine whether the bioactive peptides could have been amelogenin protein degradation products or specific amelogenin gene splice products. Thus, the splice products were prepared, and their activities were determined in vitro and in vivo. A rat incisor tooth odontoblast pulp cDNA library was screened using probes based on the peptide amino acid sequencing data. Two specific cDNAs comprised from amelogenin gene exons 2,3,4,5,6d,7 and 2,3,5,6d, 7 were identified. The corresponding recombinant proteins, designated r[A+4] (8.1 kDa) and r[A-4] (6.9 kDa), were produced. Both peptides enhanced in vitro sulfate incorporation into proteoglycan, the induction of type II collagen, and Sox9 or Cbfa1 mRNA expression. In vivo implant assays demonstrated implant mineralization accompanied by vascularization and the presence of the bone matrix proteins, BSP and BAG-75. We postulate that during tooth development these specific amelogenin gene splice products, [A+4] and [A-4], may have a role in preodontoblast maturation. The [A+4] and [A-4] may thus be tissue-specific epithelial mesenchymal signaling molecules.

Trp1, a Candidate Protein for the Store-operated Ca2+Influx Mechanism in Salivary Gland Cells

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Molecular and Functional Characterization of Protein 4.1B, a Novel Member of the Protein 4.1 Family with High Level, Focal Expression in Brain

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Cloning, Expression, and Functional Characterization of the β Regulatory Subunit of Human Methionine Adenosyltransferase (MAT II)

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.

Temporal Separation of Insulin-stimulated GLUT4/IRAP Vesicle Plasma Membrane Docking and Fusion in 3T3L1 Adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.

A Mutant Form of Vascular Endothelial Growth Factor (VEGF) That Lacks VEGF Receptor-2 Activation Retains the Ability to Induce Vascular Permeability

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

Interferon-gamma improves muscle flap microcirculation in double-strand RNA-induced inflammation.

Endothelial cell (EC) activation and subsequent expression of leukocyte adhesion molecules are initial events in multiple pathological processes. Viral double-strand ribonucleic acid (dsRNA) induces EC adhesion protein expression and leukocyte adhesion in vitro. Interferon-gamma (IFN-gamma) has been demonstrated to modulate the expression of certain adhesion proteins. The purpose of this study was to measure the inflammatory response to a viral mimetic--a synthetic dsRNA, polyinosinic-polycytidylic acid (poly-I:C)-on the microcirculation of a muscle flap in a rat model and to determine whether IFN-gamma attenuated the response. Two-stage surgery to create a cremaster muscle end-organ tube flap was performed on 18 male Sprague-Dawley rats in three groups. After intra-arterial injection into the abdominal aorta, the reagents (phosphate-buffered saline-bovine serum albumin [PBS-BSA] in groups I and II, and IFN-gamma in group III) were kept for 1 hour in this end-organ system. During the second stage at 16 hours, after injection into the penile vein (PBS-BSA in group I, poly-I:C in groups II and III), the flap was prepared for intravital microscopic measurement. The following parameters were measured: red blood cell velocity; vessel diameter; number of functional capillaries; and number of rolling, sticking, and transmigrating neutrophils and lymphocytes. Wilcoxon's rank sum test was used for statistical comparison. Poly-I:C caused a 70% increase in the main artery diameter and a 7% increase in velocity. But as a consequence of dynamic activation of leukocyte interaction, a 30% drop in functional capillary perfusion was observed. Injury to the entire vascular endothelium was confirmed by a 160% increase in transmigrating leukocytes. Treatment with IFN-gamma inhibited the poly-I:C-induced inflammation, as shown by 88%, 63%, and 85% decreases in rolling, sticking, and transmigrating leukocytes respectively, and by a 28% increase in capillary perfusion. Treating the system with IFN-gamma in advance, inhibited poly-I:C-induced inflammation, shown by marked decreases in rolling, adhering, and transmigrating leukocytes, and a notable increase in perfused capillaries. These observations reflect an inhibitory effect of IFN-gamma on leukocyte adhesion molecule expression in vascular endothelium in response to dsRNA in a muscle flap at the microcirculatory level.

The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate‐buffered saline and model food systems

Three strains of Listeria monocytogenes (NCTC 1194, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol 1(-1) phosphate-buffered saline (PBS) at pH 7.0 for up to 30 min at ambient temperature. Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied. The three strains also exhibited a wide variation in their resistance to high pressure. The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion). Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment. The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.

Characterization of the Binding of Serum Amyloid P to Laminin

Serum amyloid P (SAP) is a member of the pentraxin family. These are evolutionarily conserved proteins made up of five noncovalently bound identical subunits that are arranged in a flat pentameric disc. Although a variety of activities have been attributed to SAP and other pentraxins, their biological functions remain unclear. In humans SAP is a constitutive serum protein that is synthesized by hepatocytes. It is encoded by a single copy gene on chromosome 1. SAP is a component of all amyloid plaques and is also a normal component of a number of basement membranes including the glomerular basement membrane. The association and distribution of SAP within the glomerular basement membrane are altered or completely disrupted in a number of nephritides (e.g. Alport's Syndrome, type II membranoproliferative glomerulonephritis, and membranous glomerulonephritis). In the present study the binding of SAP to laminin was characterized. SAP binds to human laminin and merosin as well as mouse and rat laminins. The binding of SAP to mouse laminin is saturable and calcium-dependent. The Kd of this interaction is 2. 74 x 10(-7) M, with a SAP/laminin molar ratio of 1:7.1. Competition binding assays indicate that the binding of SAP to laminin is inhibited by both SAP and its analog, C-reactive protein, as well as phosphatidylethanolamine. In turbidity assays SAP enhanced the polymerization of laminin in a concentration-dependent manner. However, SAP did not alter the ability of laminin to serve as a cell adhesion substrate. Previous observations indicating that SAP binds to extracellular matrix components such as type IV collagen, proteoglycans, and fibronectin in concert with the data presented here suggest that SAP may play an important role in determining the structure of those basement membranes with which it is associated.

The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

Cryptosporidium and Giardia in Surface Waters in and around Manhattan, Kansas

Cryptosporidium spp. and Giardia spp. are intestinal parasites that can be transmitted through water contaminated by feces. Over a 16 month period, surface waters were examined in Kansas in order to measure local geographic distribution and seasonal fluctuation of oocyst and cyst populations. Cryptosporidium and/or Giardia were found in 86 percent of the locations surveyed. Between 1946 and 1980, giardiasis resulted in more cases of illness than any other known agent of waterborne disease (Lippy and Waltrip, 1984). While only two outbreaks were attributed to Giardia lamblia between 1965 and 1969, 56 outbreaks of waterborne giardiasis were reported between 1980 and 1985, demonstrating a substantial increase in the frequency, and perhaps recognition of the importance, of this flagellate. Cryptosporidium parvum, another intestinal pathogen, has recently been linked to human illness through waterborne transmission (Rose, 1988; Sterling, 1990; Smith and Rose, 1990). The disease is normally self-limiting in immunocompetent individuals, but may become life-threatening in immunocompromised individuals (Fayer et al., 1990; Reese et al., 1982; Ungar, 1990). Giardia lamblia and C. parvum exist in the environment in the form of cysts and oocysts, respectively. Despite the impact of these microorganisms on human health, little has been known about the epidemiology and prevalence of either parasite, especially C. parvum in distribution (Lippy and Waltrip, 1984). Recent outbreaks of cryptosporidiosis in New Mexico (Anonymous, 1986), Texas (D'Antonio et al., 1985), British Columbia (IsaacRenton et al., 1987), Sheffield and Great Yarmouth, England (Brown et al., 1989; Rush et al., 1987), and Georgia (Hayes et al., 1989) have suggested that this parasite may be an important cause of undiagnosed enteric disease. Recently, concentration techniques have been developed whereby both I To whom reprint requests should be addressed. This content downloaded from 207.46.13.105 on Wed, 25 May 2016 06:49:25 UTC All use subject to http://about.jstor.org/terms 102 TRANSACTIONS OF THE KANSAS ACADEMY OF SCIENCE G. lamblia and C. parvum can be detected in surface waters (Rose et al., 1985, 1986, 1988, 1989; Sterling et al., 1987; Stetzenbach et al., 1988). Surveys using these techniques have shown both G. lamblia and C. parvum to be potential sources of human disease in Arizona (Madore et al., 1987; Stetzenbach et al., 1988), Washington, and California (Ongerth and Stibbs, 1987). Since few studies have examined surface waters in the midwest United States for the presence of these pathogens, a study was conducted to determine whether Giardia and Cryptosporidium were present in significant numbers in surface waters in Kansas. MATERIALS AND METHODS Sample collection. One hundred gallon samples of water were pumped through polypropylene wound cartridge filters (Micro Wynd II, AMF/CUNO Division, Meriden, CT) at a rate of 3.3 gal/min (Musial et al., 1987; Rose et al., 1985). Filters were processed within 48 hours of collection and stored at 4?C. Samples were taken from three sites at Tuttle Creek State Park and River Pond Area, two sites at Milford Lake, the Big Blue River, and two sites on the Konza Prairie (Fig. 1). Filter elution. Filtered particles and microorganisms were removed by backflushing with 2.7 liters of 0.1% Tween 80 (Musial et al., 1987; Rose, 1988; Rose et al., 1986). Filters were teased apart and placed into eluate on a shaker for 10 min at 370C. Filter fibers were removed and the eluate concentrated by centrifugation at 1200 x g for 10 min. Cyst/oocyst extraction. Samples were washed in phosphate buffered saline (PBS), 1% sodium dodecyl sulfate (SDS), distilled water, and then 10% formalin. Samples were resuspended in 10 ml 1% SDS/1% Tween 80 and split in half. One half was layered onto 15 ml of Sheather's solution (500 g sucrose, 320 ml water, 9.7 ml liquid phenol) and the other half onto 15 ml Percoll-sucrose solution (5 ml 0.25 M sucrose with 5 ml Percoll). Each half was then centrifuged at 1200 x g and 800 x g, respectively, and the supernatants removed for assay. Cyst/oocyst quantification. Supernatants from the gradients were reconcentrated to a volume of 2.5 ml and air dried as five, 10 Aul aliquots onto microscope slides using egg albumin as an adhesive. Slides, including positive controls, were processed for indirect immunofluoresence viewing using the Hydrofluor@ combo detection assay (Hydrofluor Combo, Meridan Diagnostics, Inc.) except that 3 percent bovine serum albumin in PBS was found to be a superior blocking agent over Tween 20. Parasites on slides were then counted and original cyst/oocyst concentrations extrapolated. Efficiency studies. One hundred gallons of chlorinated tap water were seeded with known numbers of Giardia cysts or Cryptosporidium oocysts (2.5 x 105 and 1.0 x 106 cysts/oocysts, respectively). Samples were processed identically to river water samples. This content downloaded from 207.46.13.105 on Wed, 25 May 2016 06:49:25 UTC All use subject to http://about.jstor.org/terms VOLUME 94, NUMBERS 3-4 103

Optimization of procedures for separation of motile and nonmotile bull and rabbit spermatozoa with bovine serum albumin gradients

AbstractThe effect of equipment design, separatory media, and time and temperature of separation were studied. Discontinuous 4%/10% bovine serum albumin (BSA) gradients were used to isolate highly motile spermatozoa in rabbit and bull semen. For all conditions tested, motility of spermatozoa collected from the 4% BSA gradient layer (top) was less than or equal to the motility of the unseparated controls. Fractions collected from the 10% BSA gradient layer contained highly motile spermatozoa. In experiment 1, washed bull spermatozoa were diluted with phosphate‐buffered saline (PBS) containing 2% BSA or 4% BSA before being fractionated on BSA columns contained in test tubes. Inclusion of BSA in PBS tended to reduce loss of motility during washing, but the proportion of sperm recovered was highest in PBS. In experiment 2, motility and recovery of buck spermatozoa collected from the 10% BSA gradient region tended to be higher when fractionation temperature was 30°C as compared to 35°C, and motility was significantly higher when incubation time was 30 min as compared to 1 hr. The proportion of sperm recovered was unaffected by incubation time. In experiments 1 and 2, 41 of 48 separations resulted in at least one fraction containing spermatozoa with motility greater than or equal to 90%. In the third experiment, the surface area on which bull and buck spermatozoa were layered was increased by forming the 4%/10% BSA gradients in conical supports. Separation of sperm on conically shaped columns was not as effective as on cylinders. The use of cylinders to support the BSA gradients and a separation time of 30 min at 30°C is recommended.