A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

Using transplanted oyster (Crassostrea virginica) beds to improve water quality in small tidal creeks: a pilot study

The Eastern oyster, Crassostrea virginica, may improve water quality by filtering large quantities of particulate matter (both organic and inorganic) and nutrients from the overlying water column. Additionally, oyster reefs alter hydrodynamic conditions, further increasing the removal of particulate matter from the water column. This study examined the effects of small-scale oyster additions on sediment loading, chlorophyll a, nutrient concentrations, and flow in small tidal creeks. Two reefs were established in Hewletts Creek, New Hanover County, North Carolina. Total suspended solids (TSS), chlorophyll a, and ammonium were measured upstream and downstream of each created reef and in an adjacent control channel that lacked a reef. Data were collected monthly during ebb tides over a 10-month period between September 2000 and June 2001. In the first month after initial reef placement, mean TSS concentrations downstream of reef placement were slightly lower than those upstream of the reef. Although not statistically significant, TSS concentrations downstream of the reefs were less than upstream concentrations for five out of nine and five out of seven post-reef sampling months for the upland and the lower creek sites, respectively. Chlorophyll a concentrations were not significantly affected by initial reef placement (2×3 m), but were reduced substantially after reef enlargement (3×4 m) in one of the experimental creeks. Reef placement resulted in significant increases in ammonium concentrations downstream of the transplanted-reefs. In addition, deposition of feces and pseudofeces by the oysters resulted in accumulation of finer-grained materials in the treated channel relative to the control channels. Oyster filtration was most effective three hours following high tide, when the ratio of flow discharge to reef surface area was the highest. This work demonstrates that small oyster reefs established and maintained in some small tributary channels can reduce TSS and chlorophyll a concentrations and that the magnitude of the effect may vary over the course of the tidal cycle.     

A simple microtechnique for isoelectric focusing on a density gradient.

Isoelectric focusing in a density gradient can be performed on chromatographic columns of 10–15-ml volume. A polyacrylamide salt bridge was used to make electrical contact between the density gradient tube and the electrode compartment. An appropriate gradient mixer can easily be made from two 10-ml syringes. The small column permits a resolution of isoenzymes which is comparable to the separation with a commercial 110-ml column.With the described apparatus one uses ten times less Ampholine and biological sample. The time of isoelectric focusing is reduced three to four times.     

A new genus and species of fossil myliobatoid ray from the Fishburne Formation (lower Eocene/Ypresian) of Berkeley County,South Carolina,USA

Elasmobranch fossils recovered from the Fishburne Formation (lower Eocene/Ypresian) of Berkeley County, South Carolina, USA, include species from four genera of sharks and six genera of rays. Of particular interest was the recovery of multiple isolated teeth from a new genus and species of the cownosed ray family Rhinopteridae, which is the focus of this study. The unique crown morphology separates this genus and species from Rhinoptera. Eorhinoptera grabdai, gen. et sp. nov., is represented by small, bar-shaped teeth in the shape of greatly elongated hexagons. These teeth are the isolated elements of a dental plate. The holotype, with 12 wide root lobes, is the most elongated in the sample being 1 cm long and 1.5 mm wide, indicating an origin in the central region of the plate. Paratypes are less elongated, have 4–8 root lobes and are from more lateral rows. The crown is smooth and has a distinctly convex occlusal surface. Eorhinoptera is only the second genus of cow-nosed ray. Its distinctive crown morphology may have allowed it to exploit different kinds of prey than those favoured by rays that lacked convex tooth crowns.     

Population Dynamics and Description of Ptycholaimellus hibernus n. sp. (Nematoda: Chromadoridae)

Ptycholaimellus hibernus n. sp. from the muddy subtidal of North Inlet Estuary, Georgetown, South Carolina, is described and a key to the genus is provided. P. hibernus differs from all other species of Ptycholaimellus by the shape of the gubernaculum. Ptycholaimellus sp. 2 Hopper 1969 is synonymized with P. ponticus. The abundance of P. hibernus, measured over a 3-year period, is greatest from January to March, coinciding with minimal annual water temperatures (10-15 C). P. hibernus abundance was significantly (negatively) correlated with water temperature and (positively) with the depth of the anoxic sediment layer.     

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.     

Capillary chromatography-coupled mass spectrometry with column switching for rapid identification of proteins from 2-dimensional electrophoresis gels

An improved capillary liquid chromatography procedure, incorporating column switching in combination with mass spectrometry, is reported. The dual column system allows for rapid inject-to-inject cycle times to improve the speed of protein identification for proteomics applications. Full gradient elution of peptides from either of the two C18 columns can be achieved in less than 17 min while maintaining sufficient resolution for the peptides to be detected and fragmented by the mass spectrometer for protein identification. Importantly, the use of two columns for subsequent injections is reproducible and without carry-over. The limit of detection for the system is between 25 and 50 fmol per injection. This fully automated system is capable of analyzing and identifying proteins from an entire 96-well plate in about 27 h.     

Separation of oligo-RNA by reverse-phase HPLC.

A rapid and highly reproducible chromatographic technique has been developed for analysis and purification of complex mixtures of oligoribonucleotides. The method utilizes a column of microparticulate porous silica beads fully derivatized with octadecylsilyl groups. The column is eluted with gradients in acetonitrile/water/ammonium acetate pumped at pressures of 1500-300 psi. Most separations are completed in 5-15 min. with usually better than 1% reproducibility of absolute retention times and about 0.1% reproducibility of relative retention times. A single column accomplishes separations of mononucleosides, mononucleotides, and larger oligomers through at least 20-mers. The absolute detection limit is approximately 1 pmole of base though most of the analytical separations described use approximately 1 nmole. In favorable circumstances it is possible to use the analytical colums to purify approximately 1 mg of an oligonucleotide in a single 10-30 min. elution.     

Nutrient foraging traits in 10 co-occurring plant species of contrasting life forms

1 Responses to spatial heterogeneity of soil nutrients were tested in 10 plant species that differ in life form and successional status, but which co-occur in the South Carolina coastal plain. The morphological responses of the root system were tested by assessing scale (represented by root mass and root length densities), precision (preferential proliferation of roots in nutrient-rich patches compared with less fertile patches) and discrimination (ability to detect and proliferate within the richest patches when patches vary in nutrient concentration). We also investigated sensitivity (growth benefits gained as spatial heterogeneity of nutrients increases, measured as total biomass).
2 Ten individuals of each species were grown in pots under four treatments that had differing nutrient distribution but the same overall nutrient addition. Plants were harvested when roots reached pot edge.
3 We observed high variation between species in scale, precision and sensitivity. No significant discrimination responses were observed, although greatest root mass density occurred at intermediate fertility levels for all species.
4 We rejected the hypothesis that scale and precision are negatively correlated. Indeed, in herbaceous species alone, scale and precision were positively correlated.
5 Sensitivity was not closely related to precision, indicating that proliferation of roots in fertile patches does not always yield growth benefits in heterogeneous soils. Further, some sensitive species had very low precision, suggesting that a positive growth response in heterogeneous environments may be related to plasticity in physiology or root life span, rather than morphology.
6 Plant life form was not correlated with precision or sensitivity. However, scale of response was greater in herbs than in woody plants, possibly because the two life forms develop root systems at different rates.     

Screen-less expanded bed column: new approach for the recovery and purification of a malaria transmission blocking vaccine candidate from Pichia pastoris

An experimental malaria transmission blocking vaccine antigen, Pfs25H, expressed and secreted from Pichia pastoris was recovered and purified using a screenless expanded bed column equipped with a rotating fluid distribution system. This column was able to accommodate feed stock, containing 30% biomass, at a flow rate of 300–400 cm/h without affecting column stability. This capability is three times higher than the capability of the expanded bed column currently in use, which is equipped with a perforated plate fluid distribution system; this design could accommodate biomass concentrations of only up to 10%. The screen-less design did not affect the binding capacity, purification level or process yield and, therefore, shorten the process. Purified Pfs25H of 6.4 g were recovered from 37 l of Pichia pastoris culture in one step.     

Multi-analyte analysis of biological fluids with a recycling immunoaffinity column array.

A system for isolating and measuring up to 30 analytes in a single biological sample is described. The system is based on recycling a pre-labeled sample through an array of capillary immunoaffinity columns, each packed with glass beads, coated with a different antibody, thus enabling each column to isolate and extract a single analyte. Detection of the bound analytes is achieved by laser-induced fluorescence (LIF), using a laboratory-built scanning detector coupled to a fiber-optic spectrometer. The array can be regenerated up to 200 times, provided a suitable temperature is maintained. The individual immunoaffinity columns are able to bind between 2.9 and 3.6 ng of analyte, depending upon the individual column, with lower limits of detection (LOD) in the order of 1.6-2.8 pg/ml. The inter- and intra-assay coefficients of variation (CV) for all 30 columns in the array were less than 6.03+/-0.33 at analyte concentrations of 100 pg/ml. Comparison to standard enzyme-immunoassays demonstrated r(2) values in the range of 0.9151-0.9855 when analyzed by least-squares linear regression.     

Downsizing improves sensitivity 100-fold for hydrogen exchange-mass spectrometry

This paper describes a method that substantially improves the sensitivity of high-performance liquid chromatography hydrogen exchange-mass spectrometry (HPLC HX MS). The success of this method relies on using a capillary HPLC column (0.1mm IDx5cmL) to increase the sensitivity of electrospray ionization, while keeping analysis times short to minimize hydrogen/deuterium (H/D) exchange. A small, immobilized pepsin column and a capillary C18 trap were included in the capillary HPLC MS system to provide rapid digestion, peptide concentration, and desalting while maintaining slow H/D exchange conditions. To minimize the analysis time, dead volumes and capacities of all components were optimized. Fully deuterated cytochrome c and its fully deuterated peptic peptides were used to evaluate deuterium recovery at amide linkages. The deuterium recovery measured at low flow rates using this system spanned a range of 66-77% (average of 71%), which was similar to the range measured for a much larger system (67-80%, average 75%). Signal levels of most peptides for the downsized system increased by about 100-fold compared with the signal for the larger system. These results greatly strengthen the HPLC HX MS technique for studies where the quantity of protein is small.     

Improved protocol for chromatofocusing on the ProteomeLab PF2D

Beckman-Coulter has recently introduced the ProteomeLab PF2D for 2-D liquid separation of protein samples. The system features separation in the first dimension by chromatofocusing, followed by RP chromatography in the second dimension, allowing the analysis of complex proteomics samples. When used by the standard protocol, reproducibility and column life times are limited, making the use of the instrument very costly. We here present an improved protocol for chromatofocusing, which enhances column life by at least fivefold.     

Population genetics of the diamondback terrapin (Malaclemys terrapin)

We examined the population genetic structure of the diamondback terrapins (Malaclemys terrapin), within and among estuaries. Based on mark-recapture studies, these estuarine turtles have high site fidelity that is likely to make them vulnerable to local extinctions. We tested if observed site fidelity of adults would be reflected in intraestuarine population genetic structure of six highly polymorphic microsatellite loci (five tetranucleotide and one dinucleotide). No evidence was found for population structuring within the Charleston estuary nor among three different estuaries in South Carolina. We then examined four other terrapin populations from North Carolina to New York, as well as from the Florida Keys and from Texas. With increasing geographical distance, genetic differentiation increased from South Carolina through New York, but overall values were low. The dinucleotide locus contributed significantly more to the genetic differentiation of some population comparisons than any of the other loci. Interestingly, terrapins from South Carolina to New York were much more genetically similar to those from Texas (rho = 0.154) than to those from Florida (rho = 0.357). We attribute this pattern to extensive translocations of terrapins during the early 20th century to replenish diminished populations and to provide turtle farms with stocks. Terrapins collected in Texas were especially sought for shipment to the northeastern US because of their larger size. Our study indicates no population structure within or among adjacent estuaries. Thus, the mark-recapture information from adult and subadult feeding locations is a poor predictor of population genetic structure. Additionally, it appears that past human activities may have drastically altered the genetics of current populations. Finally, our data suggest that translocation of eggs or head starting of terrapins within estuaries or among adjacent estuaries is acceptable from a genetic standpoint.     

Blacktip shark Carcharhinus limbatus presence at fishing piers in South Carolina: association and environmental drivers

We tagged 12 Carcharhinus limbatus with acoustic transmitters and monitored their presence at five piers along the north-east coast of South Carolina, USA in 2016 and four piers in 2017 using acoustic receivers. Data were analysed with pier association indices (PAI), mixed models and fast Fourier transformation analyses to identify potential factors related to residence time and presence at piers and any cyclical patterns in visits to piers. While the majority of monitored C. limbatus were infrequently detected at piers, three (25.0%) were highly associated with piers (PAI ≥ 0.50). Of the C. limbatus that were detected after initial capture, three (25.0%) recorded detection events only at the pier where they were tagged and two individuals (16.7%) recorded at least one detection event at all monitored piers. The best-fit model explaining C. limbatus residence time at piers included terms for pier location and diel cycle (w_i = 0.88), whereas the best fit model explaining presence–absence of C. limbatus at piers included terms for tidal height, diel cycle, barometric pressure and angler count (w_i = 0.98). Carcharhinus limbatus did not appear to display cyclical patterns in their visits to piers. Along the north-east coast of South Carolina, association of C. limbatus with piers is a phenomenon for a proportion of mature individuals, but continued research is necessary to understand if this behaviour is driven by attraction to and feeding on angler discards or increased foraging opportunities resulting from the attraction of potential prey to the physical structure provided by piers.     

Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt(+2) immobilized metal affinity chromatography

Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.     

Binding study of semotiadil and levosemotiadil with alpha(1)-acid glycoprotein using high-performance frontal analysis.

High-performance frontal analysis (HPFA) was used to investigate the binding properties of human alpha(1)-acid glycoprotein (AGP) with semotiadil ((R)-isomer, Ca-channel blocker) and its antipode levosemotiadil ((S)-isomer, Ca- and Na-channel blockers). An on-line HPLC system consisting of a HPFA column, an extraction column, and an analytical HPLC column was used to determine the unbound concentrations of these enantiomers, and the experimental data were subsequently subjected to the Scatchard analyses to estimate their binding parameters. The binding affinity of the (R)-isomer (K = 3.17 x 10(7) M, n = 0.74) is approximately 1.2 times stronger than that of (S)-isomer (K = 2.59 x 10(7) M, n = 0.74). An enantioselective competitive binding study indicated that both enantiomers are bound at the same site on AGP molecules.     

A pulsing device for packed-bed bioreactors: I. Hydrodynamic behaviour

This paper describes a new pulsing device which permits the insertion in pulsing form of a liquid phase fed into an equipment where a microbial or enzymatic transformation occurs. It also analyzes the modifications of the flow model caused by the pulsation generated by means of three kinds of pulsators: A hydropneumatic pulsator, a selfpropelled pulsator and a newly designed elastic membrane pulsator.The hydrodynamic behaviour of a packed-bed column, to which each of these pulsators has been connected is compared with the correspondent system without pulsation. The flow model is determined by the study of the curves of residence times distribution, obtained by using a stimulus-response technique. A computer programme has been used to determine the axial dispersion coefficients from the response curves. In all cases we worked within a wide range of Re _p(10–215).     

Inability of gestational hormones to account for the inhibitory effects of pregnancy plasmas on lymphocyte responses in vitro.

Plasma from pregnant women has a marked inhibitory effect on lymphocyte responses in vitro. While much evidence suggests that this is due to an immunologic mechanism, an apparent lack of specificity and the known suppressive effects of several hormones on immune function has led to speculation that the inhibitory effects could be due to increased concentrations of gestational hormones. We have investigated the effects of a wide range of concentrations of estrone, estradiol, estriol, progesterone, human chorionic gonadotropin (HCG), and hydrocortisone on lymphocyte responses to mitogens and allogeneic cells. None of these hormones were capable of inhibiting lymphocyte DNA synthesis even at concentrations several times the maximum physiologic plasma levels occurring during pregnancy. Very high, supraphysiologic concentrations were found to be inhibitory. In investigating the mechanism of the hormonal inhibition we found that if they were removed from the media at various times after initiation of culture, the estradiol, HCG, and to a lesser extent the hydrocortisone effects were all reversible. Estradiol and HCG differed from hydrocortisone in that the former were inhibitory only when added at the initiation of culture, whereas hydrocortisone was inhibitory even when added 24 hr later. In summary, while extremely high concentrations of gestational hormones are inhibitory, the quantities which occur physiologically in gestational plasmas are not able to suppress lymphocyte responses and thus cannot account for their inhibitory effects.