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Query: UNIPROT:P30536 (PBS)
 9,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subunit structure of soluble goat hepatic lectin was studied by determining molecular weight under nondenaturing conditions by gel filtration and denaturing conditions by SDS PAGE. Affinity purified lectin was subjected to HPLC on asahipack column equilibrated with 10 mM Tris-HCl pH 7.5, containing 1 mM CaCl2, 1mM 2-mercaptoethanol and 0.1M NaCl. The lectin was eluted under single peak at retention time of 12 min. corresponding to molecular weight of 38Kd. On SDS-PAGE in the presence and absence of 2-mercaptoethanol protein moved as single band with Rm 0.45, which corresponds to molecular weight of 20 Kd. The results suggest that soluble goat hepatic lectin is a dimmer of identical subunits which are linked together by noncovalent interactions. The interaction of monoclonal antibodies raised against soluble goat hepatic lectin (MGHL/20) with hemagglutinin from different species as sheep, human, rat, bovine and chicken was studied in PBS by solid phase binding assay. MGHL/20 showed 29.89% binding with these lectin. However no binding was found with Ca++ dependent membrane bound lectin. These results indicate that soluble goat hepatic lectin possesses antigenic structural relationship with soluble 14 K lectin family.
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PMID:Subunit structure of Ca++ dependent soluble goat hepatic lectin: evidence that it has antigenic structural relationship with soluble 14K lectin family. 886 13

The effect of ionic strength and pH of carrier solutions on the separation of liposomes by flow field-flow fractionation (flow FFF) has been studied for the determination of accurate vesicle size distribution of liposomes. Retention behaviors of liposomes (PC/PG/cholesterol) are observed in typical buffer solutions (PBS and Tris-HCl) of various ionic strengths as carrier liquids in flow FFF. The average diameters of collected fractions at each flow FFF run are measured by photon correlation spectroscopy (PCS) for the comparison with FFF calculations at corresponding time interval of collected fractions. A reasonable separation of liposomes is observed at I = 0.016 M for both buffer solutions. Retention of liposomes is found to be elongated at ionic strengths higher than an optimum condition found experimentally, but it is shortened at a lower ionic strength due to the electrostatic interaction between the channel wall and the liposomes. Finally, size distributions of liposomes are provided comparing the liposome preparations by flow FFF.
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PMID:Size characterization of liposomes by flow field-flow fractionation and photon correlation spectroscopy. Effect of ionic strength and pH of carrier solutions. 969 17

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (-3, -10, and -50 degrees C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at -10 degrees C/min (P<0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P<0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk-raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk-raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P>0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of -10 degrees C/min preserves the motility and fertilization capacity of mouse spermatozoa.
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PMID:Fertilizing potential of mouse spermatozoa cryopreserved in a medium containing whole eggs. 1067 48

Protocols for the successful manipulation and preservation of semen in a given species depend upon a fundamental knowledge of how spermatozoa respond to the physicochemical conditions of the extension media; methods developed for the preservation of eutherian spermatozoa may not necessarily be suitable for marsupial semen. The aim of this study was to investigate the effects on koala sperm motility of serial dilution, changes in temperature, diluent pH and osmolality to establish the optimal physicochemical conditions for short-term semen storage. This study showed that electroejaculated koala semen diluted 1&ratio;1 (v/v) with PBS frequently coagulated after incubation at 35 degrees C, but that further dilution and incubation resulted in a corresponding increase in the percentage of spermatozoa swimming in a non-linear trajectory. The effect of rapid temperature change on the motility of koala spermatozoa was investigated by exposing semen, initially diluted at 35 degrees C, to temperatures of 45, 25, 15 and 5 degrees C. Although sperm motility was reduced after incubation at 45 degrees C, a rapid decrease in temperature of up to 20 degrees C did not result in a significant reduction in sperm motility. However, contrary to evidence in other marsupials, there was a small but significant decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 degrees C. The effects of diluent pH and osmolality on the motility of koala spermatozoa were investigated. These experiments indicated that diluents for koala sperm manipulation should buffer in a pH range of 7-8 and have an osmolality of approximately 300 mmol kg(-1). The final experiment compared the relative effectiveness of Tris-citrate buffer (1% glucose) and PBS to maintain koala sperm motility over a range of incubation temperatures (5-35 degrees C) for up to 8 days. Reduction in sperm motility was directly related to temperature, and motility was sustained for the longest duration when stored at 5 degrees C. The Tris-citrate buffer solution was superior to PBS as a preservation diluent at all temperatures, and koala spermatozoa remained motile even after 42 days storage at 5 degrees C. Spermatozoa diluted in PBS (with Ca(2+) or Mg(2+)) and cooled to 5 degrees C showed evidence of an unusual motility pattern, similar to that of hyperactivated eutherian spermatozoa. This study showed that koala spermatozoa respond to different physicochemical conditions associated with short-term liquid storage in essentially the same way as the spermatozoa of eutherian mammals, although koala spermatozoa appear to be more tolerant of rapid temperature shock. The results of this study can be used to make informed selections with regard to appropriate diluent composition and improved short-term sperm preservation protocols and represent the first such database for any species of marsupial.
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PMID:Optimal physicochemical conditions for the manipulation and short-term preservation of koala (Phascolarctos cinereus) spermatozoa. 1086 91

A capillary-column-based bioseparator/bioreactor was developed for detection of Escherichia coli O157:H7 by chemically immobilizing anti-E. coli O157:H7 antibodies onto the inner wall of the column, forming the "sandwich" immunocomplexes (immobilized antibody-E. coli O157: H7-enzyme-labeled antibody) after the sample and the enzyme-labeled antibody passed through the column and detecting the absorbance of the product in the bioreactor with an optical detector. The effects of the blocking agent, flow rate of samples and substrates, buffer, MgCl2, and pH on the detection of E. coli O157:H7 were investigated. The parameters, 2% BSA in 1.0 x 10-2 M, pH 7.4, PBS as the blocking agent, 0.5 mL/h as the sample flow rate, 1.0 x 10(-2) M MgCl2, and 2.0 x 10(-4) M p-nitrophenyl phosphate in 1.0 M, pH 9.0 Tris buffer as the substrate for the enzymatic reaction, and 1.0 mL/h as the substrate flow rate, were used in the bioseparator/bioreactor system for detection of E. coli O157:H7. The selectivity of the system was checked, and other pathogens, including Salmonella typhimurium, Campylobacterjejuni, and Listeria monocytogenes, had no interference with the detection of E. coli O157:H7. Its working range was from 5.0 x 10(2) to 5.0 x 10(6) cfu/mL, and the total assay time was < 1.5 h without any enrichment. The relative standard deviation was approximately 2.0-7.3%.
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PMID:An antibody-immobilized capillary column as a bioseparator/bioreactor for detection of Escherichia coil O157:H7 with absorbance measurement. 1172 16

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.
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PMID:Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement. 1222 97

In the present study PCR was applied to detect leptospires in human urine. Several approaches for sample processing were evaluated to optimize the detection of leptospires in urine mixed with this bacterium. Furthermore, some changes in the composition of the reaction mix were studied. No amplification was observed in acidic urine, therefore neutralization of the sample immediately after collection is strongly recommended. PBS gave better results than Tris or NaOH as neutralizing reagents. Freezing and thawing of samples before processing yielded negative results. Elimination of epithelial cells, leukocytes and crystals by centrifugation at 3,000 rpm at room temperature increased sensitivity. In addition, both the washing step after collecting leptospires by centrifugation and the inclusion of 0.1% bovine serum albumin in the reaction mix minimized the interference of other inhibitory compounds. These modifications were useful to improve the detection of Leptospira in urine by PCR.
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PMID:Recommendations for the detection of Leptospira in urine by PCR. 1509 96

Multilayer films consisting of polyethylenimine (PEI) and albumin were successfully prepared on biomedical 316L stainless steel surface via electrostatic self-assembly of the PEI and albumin. The process of electrostatic self-assembly of PEI/albumin was monitored by125I radiolabeling, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The EIS data revealed that the multilayer coating was stable in Tris-HCl (pH 7.35) buffer solution for 21 days. 125I radiolabeling experiments indicated that less than 10% albumin was eluted by PBS in 45 days. Static platelet adhesion experiments indicated that the PEI/albumin deposited on stainless steel could resist platelet adhesion effectively. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.
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PMID:Fabrication of alternating polycation and albumin multilayer coating onto stainless steel by electrostatic layer-by-layer adsorption. 1526 Oct 73

FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.
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PMID:1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins. 1576 96

A study was undertaken to determine the effects of oviductal proteins obtained from various stages of the estrous cycle on spermatozoa characteristics in buffaloes. Oviducts were collected from apparently healthy buffalo genital tracts (nonluteal and luteal stage of estrous cycle) and separated into isthmus and ampulla. Each segment of oviduct (nonluteal and luteal) was flushed with PBS (pH 7.4). The flushing obtained was centrifuged (3000 rpm; 30 min), filtered (0.2 microm) and frozen at -20 degrees C. The proteins in pooled nonluteal isthmic and ampullary and luteal isthmic and ampullary fluids were precipitated overnight using ammonium sulphate, centrifuged (10000 rpm; 30 min) and dialyzed (>10 kDa). After protein estimation, aliquots of samples containing 10 mg proteins were lyophilized in cryovials and stored in frozen form at -20 degrees C. Six pooled good-quality ejaculates collected by artificial vagina method from two Murrah buffalo bulls were utilized for the study. After fresh semen analysis, each pooled ejaculate was splited into five parts and extended in Tris-egg yolk-citrate extender (20% egg yolk; 7% glycerol), so that final dilution yielded approximately 60 million sperm cells per ml, and cryopreserved in 0.5 ml French straws (30 million sperm cells/straw) in LN(2) (-196 degrees C). Before freezing, nonluteal isthmic and ampullary and luteal isthmic and ampullary proteins were incorporated at the rate of 1mg/ml of extended semen. The equilibrated and frozen-thawed (37 degrees C for 30 s) semen was evaluated for motility, live %, acrosomal integrity percentage, bovine cervical mucus penetration test and hypo-osmotic sperm swelling test. Besides this, spermatozoa from treatment and control groups were incubated at 37 degrees C for 6 h in sperm TALP. Among the nonluteal and luteal oviductal proteins, the former maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity than the control group. Between the isthmic and ampullary proteins, the isthmic proteins incorporated group maintained higher (P < 0.05) post-thaw sperm motility, live %, and acrosomal integrity. Similarly, higher sperm penetration distance in cervical mucus was recorded in nonluteal isthmic proteins incorporated group. But, irrespective of the stage of an estrous cycle, isthmic proteins included group maintains higher sperm membrane integrity as revealed by higher (P < 0.05) swollen sperm percentage in response to hypo-osmotic solution than the ampullary proteins included and control groups. Similarly, at any time during incubation the sperm motility and viability was higher (P < 0.05) in isthmic proteins treated group than the ampullary and control group. But, the same trend was not observed in terms of acrosomal integrity percentages. It is inferred that inclusion of oviductal proteins in the extender prior to freezing improved post-thaw semen quality. Oviductal proteins differentially affected sperm function depending upon the region of oviduct and the stage of estrous cycle at which the proteins were obtained.
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PMID:Modulation of post-thaw sperm functions with oviductal proteins in buffaloes. 1595 Apr 8


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