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PubMed1990

Refolding and reassembly of separate alpha and beta chains of class II molecules of the major histocompatibility complex leads to increased peptide-binding capacity.

Class II molecules of the major histocompatibility complex present antigenic peptides to helper T cells. These are heterodimeric glycoproteins consisting of one alpha and one beta chain. Two different alpha/beta heterodimeric conformations as well as the separate alpha and beta chains bind specific peptides. The alpha chain is thought to have one and the beta chain two intramolecular disulfide bonds. In the present study we have reduced these disulfide bonds in the murine major histocompatibility complex molecule I-Ad, which led to the release of bound peptides from all conformations and to unfolding of the separate chains. The separate alpha and beta chains could be refolded to their native structure by reoxidation of the cysteines. Refolding was accompanied by reassembly of the separated chains to the alpha/beta heterodimer. Both the separated alpha and beta chains and the alpha/beta heterodimer bound significantly higher amounts of antigenic peptide after reduction and reoxidation, as compared to the untreated protein.

Animals↗

PubMed1997

Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

Adenosine Triphosphatases↗

PubMed1998

Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.

Hepatitis C virus (HCV) helicase catalyzes the ATP-dependent strand separation of duplex RNA and DNA containing a 3' single-stranded tail. Equilibrium and velocity sedimentation centrifugation experiments demonstrated that the enzyme was monomeric in the presence of DNA and ATP analogues. Steady-state and pre-steady-state kinetics for helicase activity were monitored by the fluorescence changes associated with strand separation of F21:HF31 that was formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a complementary 3'-fluorescein-tagged 21-mer (F21). kcat for this reaction was 0.12 s-1. The fluorescence change associated with strand separation of F21:HF31 by excess enzyme and ATP was a biphasic process. The time course of the early phase (duplex unwinding) suggested only a few base pairs ( approximately 2) were disrupted concertedly. The maximal value of the rate constant (keff) describing the late phase of the reaction (strand separation) was 0. 5 s-1, which was 4-fold greater than kcat. Release of HF31 from E. HF31 in the presence of ATP (0.21 s-1) was the major contributor to kcat. At saturating ATP and competitor DNA concentrations, the enzyme unwound 44% of F21:HF31 that was initially bound to the enzyme (low processivity). These results are consistent with a passive mechanism for strand separation of F21:HF31 by HCV helicase.

Adenosine Triphosphate↗

PubMed2004

GPI7 involved in glycosylphosphatidylinositol biosynthesis is essential for yeast cell separation.

GPI7 is involved in adding ethanolaminephosphate to the second mannose in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. We isolated gpi7 mutants, which have defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature. WSC1, RHO2, ROM2, GFA1, and CDC5 genes were isolated as multicopy suppressors of gpi7-2 mutant. Multicopy suppressors could suppress the growth defect of gpi7 mutants but not the cell separation defect. Loss of function mutations of genes involved in the Cbk1p-Ace2p pathway, which activates the expression of daughter-specific genes for cell separation after cytokinesis, bypassed the temperature-sensitive growth defect of gpi7 mutants. Furthermore, deletion of EGT2, one of the genes controlled by Ace2p and encoding a GPI-anchored protein required for cell separation, ameliorated the temperature sensitivity of the gpi7 mutant. In this mutant, Egt2p was displaced from the septal region to the cell cortex, indicating that GPI7 plays an important role in cell separation via the GPI-based modification of daughter-specific proteins in S. cerevisiae.

Amino Acid Sequence↗

PubMed2006

Expanding the subproteome of the inner mitochondria using protein separation technologies: one- and two-dimensional liquid chromatography and two-dimensional gel electrophoresis.

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the alpha-subunit of F1F0 ATP synthase that differed due to a change in pI or hydrophobicity.

Animals↗

PubMed1999

HPCPC separation of proteins using polyethylene glycol-potassium phosphate aqueous two-phase.

High Performance Centrifugal Partition Chromatography (HPCPC) is a practical and suitable method, particularly on the preparative scale, for the separation of biomolecules such as proteins, enzymes, etc. Aqueous two-phase system is also very attractive for the isolation of biomolecules. Aqueous polymer phase system composed of polyethylene glycol 6000-potassium phosphate has been used for the countercurrent chromatographic separation of bovine serum albumin (BSA) and lysozyme using HPCPC. The separation of BSA and lysozyme under various conditions such as various flow rates, rotational speeds, pH of the solvent, and the retention of stationary phase has been studied in the present investigation. The baseline separation of BSA and lysozyme has been also observed. The results of this study demonstrate that HPCPC is useful for separation of proteins with aqueous two-phase systems.

Chromatography, Liquid↗

PubMed2003

The separation of codeine from nonprescription combination analgesic products.

In the United Kingdom number of products containing codeine in combination with nonopioid analgesics are available from pharmacies for purchase without a prescription. These products may be abused and it has been reported that in order to avoid nonopioid toxicity, some users may attempt to separate the components of combination analgesics, by mixing the dosage form in water and passing it through a coffee filtration apparatus (coffee filters). The present research sought to test the ease of separation of products available in the United Kingdom. The results obtained indicated that separation was possible; however, the extent of separation was influenced by the volume of water used and the product chosen. Healthcare professionals must be aware that separation may be attempted by those wishing to abuse codeine; however, significant nonopioid toxicity could still arise in the abuser.

Analgesics↗

PubMed1981

Mechanics of chromosome separation during mitosis in Fusarium (Fungi imperfecti): new evidence from ultrastructural and laser microbeam experiments.

The anaphase-telophase spindle usually elongates, and it has been assumed that the spindle pushes the incipient daughter nuclei apart. To test this assumption, we used a laser microbeam to sever the central spindle of the fungus, Fusarium solani, and measured the rate of separation of incipient daughter nuclei. When the microbeam was aimed beside the spindle separation occurred at a rate (8.6 micrometer/min) that did not differ significantly from the rate (7.6 micrometer/m) in unirradiated cells. But when the spindle was irradiated, it broke, and the separation was much faster (22.4 micrometer/min). Irradiation of cytoplasm lateral to one spindle pole resulted in a 1.5 micrometer/min reduction in the rate (6.1 micrometer/min) of separation. From these and other data, we infer that extranuclear forces, presumably involving astral microtubules, pull on the incipient daughter nuclei and that the central spindle limits the separation rate. Astral microtubules are associated with the plasma membrane or, sometimes, with the rough endoplasmic reticulum. Most of the spindle microtubules that are present at metaphase are depolymerized during anaphase and early telophase.

Anaphase↗

PubMed2003

Preparation of monoclonal antibodies using the electrophoresis separation instrument, Gradiflow.

Gradiflow, a preparative electrophoresis separation device, was utilized to develop and test generic protocols for the preparation of monoclonal antibodies (MAbs) from tissue culture supernatant and ascites fluid. The charge based protocol separated the high pI antibodies from the lower isoelectric points (pI) contaminants by either moving the antibody (ascites fluid) or contaminants (tissue culture supernatant) through a polyacrylamide separation membrane. A total of 60 separations were performed with tissue culture supernatant, and a further 30 separations with ascites fluid. The Gradiflow procedure resulted in higher yields, equivalent functionality and similar purity compared with affinity chromatography antibody preparation on protein A and G. The results suggest that the Gradiflow protocols may be an alternative method of antibody preparation for these samples.

Animals↗

PubMed1999

Separation anxiety in children and adolescents treated with risperidone.

Risperidone is an atypical antipsychotic that has been investigated as a treatment for several severe psychiatric disorders in children and adults. A low dose of risperidone was added to other medications to treat two adolescent boys with obsessive compulsive disorder (OCD) and a childhood history of separation anxiety disorder, and one prepubertal boy with a history of attention deficit hyperactivity disorder and escalating behavior suggestive of mania. Each patient developed severe separation anxiety that resolved when risperidone was discontinued. The response was similar to that described in Tourette's disorder patients treated with haloperidol and pimozide. Two of the patients were treated subsequently with olazapine without a recurrence of separation anxiety. We discuss the diagnostic issues concerning separation anxiety induced by antipsychotic medications, and suggest that the atypical antipsychotics may differ in their tendency to elicit separation anxiety.

Adolescent↗

PubMed1999

Cerebral near infrared spectroscopy: emitter-detector separation must be increased.

We have compared the effect of increasing optode separation (range 0.7-5.5 cm) on the sensitivity of near infrared spectroscopy (NIRS) to discrete reductions in scalp and cerebral oxygenation in 10 healthy men (mean age 32, range 26-39 yr) using multichannel NIRS. During cerebral oligaemia (a mean reduction in middle cerebral artery flow velocity of 47%) induced by a mean reduction in end-tidal PCO2 of 2.4 kPa, the decrease in oxyhaemoglobin detected by NIRS became significantly greater with increasing optode separation (P < 0.0001). In response to scalp hyperaemia induced by inflation and release of a pneumatic scalp tourniquet, increases in oxyhaemoglobin became significantly smaller with increasing optode separation (P < 0.0002). These results are consistent with theoretical models of the behaviour of NIR light in the adult head and support the concept of using multi-detector NIRS to separate intra- and extracranial NIR signal changes. However, the emitter-detector separation used by currently available cerebral oximeters is not large enough to provide optimal spatial resolution.

Adult↗

PubMed2006

The craniopagus malformation: classification and implications for surgical separation.

Craniopagus twins (CPT) are an uncommon, highly fascinating accident of nature. The clinical pathology of this complex entity is reviewed and placed in perspective. A logical classification aids understanding of the anomaly, and is essential to gauge outcome from separation attempts. 'Partial forms' lack significant shared dural venous sinuses (SDVS) and 'Total forms' with SDVS also exhibit more severe compressional brain distortion. Our classification consists of Partial Angular (PA), Partial Vertical (PV), Total Angular (TA) and Total Vertical (TV, formerly O'Connell Types I-III). Total vertical has a continuous cranium, and inter-twin axial facial rotation <40 degrees (Type I), 140-180 degrees (Type II) or intermediate (Type III). The term 'Angular' denotes an inter-twin longitudinal angle below 140 degrees , regardless of axial rotation. Our review categorized 64 well-delineated CPT, including 41 operative separation attempts in small children since initial success in 1952. Just over one-half were TV, almost one-third TA, and partial forms accounted for the remaining one-sixth. About 30% of CPT had shared or fused brain tissue, and a similar percentage of TA twins shared a posterior fossa. Partial forms had significantly higher birth weights, were separated at an earlier age (6 versus 11 months) and had lower mortality and better outcome compared with Total forms. A multi-staged surgical separation for Total CPT had a significantly better mortality than single-staged separation. Discussion emphasizes embryological, anatomical and clinical aspects of the malformation, with emphasis upon obstacles to a successful outcome.

Brain↗

PubMed1976

Separation of benefin and trifluralin by gas chromatography using a liquid crystal column.

Benefin (N-[n-butyl]-N-ethyl-2,6-dinitro-alpha, alpha, alpha-trifluoro-p-toluidine) and trifluralin (2,6-dinitro-N,N-di-n-propyl-alpha, alpha, alpha-trifluoro-p-toluidine) (Figure 1) are isomeric preemergent herbicides (1,2). As they possess different activities, their use necessitates the development of a routine analytical method which differentiates between them. The separation of these isomers by gas-liquid chromatography (GLC) using conventional column packings has proved extremely difficult. Two partly successful separations have been previously reported to date. A partial separation was obtained using a Durapak-Carbowax 400/Poracil C column (3) and a complete separation using a column packed with a monomolecular layer of Carbowax 20M on Chromosorb W (4). Unfortunately, this latter column has a very limited life and the analysis time is 25 minutes. This paper reports a rapid and reproducible separation of benefin and trifluralin using a liquid crystal stationary phase.

Chromatography, Gas↗

PubMed2001

Optimization strategies and modeling of separations of dansyl-amino acids by cyclodextrin-modified capillary electrophoresis.

Presented in this study is an approach to optimize conditions for capillary electrophoresis separations of multianalyte enantiomeric pairs (D- and L-dansyl (Dns)-amino acids) that involves the rational use of combinations of cyclodextrins (CDs) as enantioselective running buffer additives. Migration data is experimentally obtained for a range of concentrations for native CDs used individually and employed to determine inclusion constants for the Dns-amino acids of interest. An expression for the mobility of the amino acids when multiple (two in this work) CDs are present in the running buffer is used to simulate separations for more complex CD systems. A chromatographic response function involving predicted resolution is generated to gauge the quality of these separations. Simplex methods are then employed for the first time to optimize conditions for the separation of amino acid enantiomers. The validity of this approach is demonstrated for separations of five Dns-amino acid enantiomers using gamma- and beta-CDs at various concentrations. Extending the dual-CD approach to other CDs and increasing the number of CDs beyond two should be possible. To this end, preliminary experiments are performed by using several available single-isomer, derivatized CDs (individually) to determine if they have potential for further studies. Although results with these particular derivatized CDs are not encouraging, we did find that molecular mechanics modeling is useful in interpreting those cases in which low inclusion constants possibly contributed to the ineffectiveness of the CDs.

Amino Acids↗

PubMed1986

Precocious meiotic centromere separation of a novel yeast chromosome.

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.

Centromere↗

PubMed1996

Separation of bacterial capsular and lipopolysaccharides by preparative electrophoresis.

Preparative gel electrophoresis was used to separate and purify extracellular, capsular and lipopolysaccharides (EPSs, CPSs, and LPSs, respectively) from crude bacterial extracts. The procedure effectively separates CPS from LPSs. In addition discreet size ranges of these various polysaccharides can be isolated. The 'rough' (R-type), 'smooth' (S-type), and 'semi-smooth' LPSs were separated from one another. In addition different size classes of 'semi-smooth', or S-type LPS, can be separated. This procedure was demonstrated for diverse bacterial species, including the soil bacteria Rhizobium fredii, and the enteric bacterial species, Salmonella enteritidis and Proteus mirabilis. In the latter case, it was also possible to separate capsular polysaccharide from its lipid-bound form.

Electrophoresis, Polyacrylamide Gel↗

PubMed2003

Impact of separating drug prescribing and dispensing on provider behaviour: Taiwan's experience.

In many Asian countries, physicians both prescribe and dispense drugs. This practice is hypothesized to have caused high drug expenditure and widespread prescription of antibiotics in Asia. Recently, Taiwan implemented the separation policy on an experimental basis. This paper's objective is to empirically evaluate the impact of Taiwan's reform to separate drug prescribing and dispensing on drug expenditure and total health expenditure. The research design consists of a pre/post comparison of the experimental with the control sites (difference-in-difference). Separation policy was implemented in Kaohsung and Taipei in March 1997, and expanded to Chia-yi and Taichung in March 1998. Changes in drug prescription behaviour before and after implementation in these two pairs of experimental cities were compared to Hsin-chu and Tainan (control), where separation policy was not implemented during the study period. To reduce resistance, providers in experimental sites were allowed to hire on-site pharmacists and dispense drugs through them if they chose to do so. Our study sample consists of all outpatient visits to clinics in the study sites between December 1996 and June 1998, with a total of 55.23 million claim records. The drug prescription rate, drug expenditure and total health expenditure per visit were the main outcome measures. We found that the probability of prescription and drug expenditure per visit were, respectively, 17-34% and 12-36% less among visits to clinics without on-site pharmacists, compared with the control sites. However, no difference in total health expenditure was found between these two types of visits. Hence, the separation policy could be effective in reducing drug expenditure and affecting prescription behaviour, but is less certain as a policy for reducing total health expenditure. We also found that the policy has practically no effect on clinics that have on-site pharmacists.

Drug Costs↗

PubMed1994

Rapid pulsed field separation of DNA molecules up to 250 kb.

Pulsed field gel electrophoresis (PFGE) is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kilobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90 degrees to be effective. However, reorientation angles of 90 degrees and even less will resolve DNA fragments a few hundred kb and smaller approximately 5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90 degrees reorientation angles is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120 degrees. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large insert bacterial clones, such as those using cosmid, Fosmid, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.

DNA, Bacterial↗