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Biomedical subjects

S L Cohen

Publications and source records attributed to S L Cohen.

At least 19 recordsLinked to original sources
PubMed2000

Mass spectrometric analysis of mercury incorporation into proteins for X-ray diffraction phase determination.

Heavy-atom incorporation is an essential and often rate-limiting step in the determination of phases for X-ray diffraction studies of protein structures. Until the present, there has been no practical method (short of the X-ray diffraction experiment itself) to judge the success and extent of incorporation. Here we show that mass spectrometry is an effective tool for determining the extent of heavy-atom incorporation in proteins. In particular, we demonstrate the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) for assaying mercury derivatization of cysteinyl thiol groups in proteins. Each of these mass spectrometric methods has advantages and drawbacks. ESI-MS provides a more accurate quantitative measurement of the extent of mercury incorporation, while MALDI-MS provides a useful lower limit to the level of mercury incorporation. Conversely, MALDI-MS does not require removal of excess derivatization reagents, salts and buffers, thus permitting facile analysis of single protein crystals as well as rapid, semiquantitative evaluation of the extent of protein mercuration. The approaches described in the present paper have contributed to the successful X-ray analyses of several noteworthy protein structures.

Crystallization↗
PubMed1998

Crystal structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain.

The HERG voltage-dependent K+ channel plays a role in cardiac electrical excitability, and when defective, it underlies one form of the long QT syndrome. We have determined the crystal structure of the HERG K+ channel N-terminal domain and studied its role as a modifier of gating using electrophysiological methods. The domain is similar in structure to a bacterial light sensor photoactive yellow protein and provides the first three-dimensional model of a eukaryotic PAS domain. Scanning mutagenesis of the domain surface has allowed the identification of a hydrophobic "hot spot" forming a putative interface with the body of the K+ channel to which it tightly binds. The presence of the domain attached to the channel slows the rate of deactivation. Given the roles of PAS domains in biology, we propose that the HERG N-terminal domain has a regulatory function.

Animals↗
PubMed1998

Structural conservation in prokaryotic and eukaryotic potassium channels.

Toxins from scorpion venom interact with potassium channels. Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry. One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding. The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels. This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.

Amino Acid Sequence↗
PubMed1998

The structure of the potassium channel: molecular basis of K+ conduction and selectivity.

The potassium channel from Streptomyces lividans is an integral membrane protein with sequence similarity to all known K+ channels, particularly in the pore region. X-ray analysis with data to 3.2 angstroms reveals that four identical subunits create an inverted teepee, or cone, cradling the selectivity filter of the pore in its outer end. The narrow selectivity filter is only 12 angstroms long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at the center of the bilayer. Main chain carbonyl oxygen atoms from the K+ channel signature sequence line the selectivity filter, which is held open by structural constraints to coordinate K+ ions but not smaller Na+ ions. The selectivity filter contains two K+ ions about 7.5 angstroms apart. This configuration promotes ion conduction by exploiting electrostatic repulsive forces to overcome attractive forces between K+ ions and the selectivity filter. The architecture of the pore establishes the physical principles underlying selective K+ conduction.

Amino Acid Sequence↗
PubMed1998

Behavioral momentum: the effects of the temporal separation of rates of reinforcement.

In Part 1 of the experiment, rats responded under a variable-interval (VI) 30-s schedule and a VI 120-s schedule, with each in effect for a block of consecutive sessions. That is, the two VI schedules were presented in successive conditions. In Part 2 the VI schedules alternated each day, and in Part 3 the schedules alternated within the session as a multiple schedule. For half of the rats in Parts 1 and 2, the VI schedule alternated every few minutes within the session with a stimulus that signaled extinction. For each part, once response rates had stabilized, resistance to change was measured by prefeeding and extinction. When the schedules were examined in successive conditions (Part 1), resistance to extinction was greater under the VI 120-s schedule of reinforcement than under the VI 30-s schedule, but no consistent differences in resistance to prefeeding were observed between the two VI schedules. When the VI schedules alternated each day (Part 2), resistance to extinction was greater under the VI 120-s schedule. However, no consistent differences in resistance to prefeeding were observed between the VI schedules without extinction in Group A, but resistance to prefeeding was greater under the VI 30-s schedule for rats with the added extinction component in Group B. When the VI schedules alternated within the session as a multiple schedule (Part 3), resistance to extinction and resistance to prefeeding were greater under the VI 30-s schedule. The data suggest that different rates of reinforcement, and their accompanying discriminative stimuli, must be compared within the same session (or at least on alternate days) to produce data consistent with the behavioral momentum model.

Animals↗
PubMed1997

Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

In this report we describe a novel approach to the mass spectrometric analysis of whole proteins from gels. The strategy consists of three components: conventional SDS-PAGE gels, reversible negative staining procedures, and passive elution of proteins from gels followed by mass spectrometric analysis. Protein bands are excised from SDS-PAGE gels, destained, and extracted. For gel loadings > or = 25 pmol of soluble protein, the proteins can be directly extracted into a solution consisting of formic acid/water/2-propanol. The recovered protein is suitable for matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization mass spectrometric analysis. For gel loadings < 25 pmol protein, the mass spectrometric response, using the direct extraction procedure, drops off sharply, an outcome that is attributed to protein recovery losses. To offset the protein losses, the extraction procedure is slightly modified by performing the passive extraction of the gel with a saturated MALDI matrix solution. During the extraction period, the matrix is allowed to crystallize, forming a suspension in solution. Protein that elutes from the gel has a chance to cocrystallize with the matrix that can be retrieved for MALDI-MS analysis. This method of "capturing" eluted protein into matrix crystals is sensitive to 1 pmol of recombinant mouse leptin protein (16 kDa) loaded onto SDS-PAGE gels and can be used for proteins as large as 70 kDa. Our strategy has particular application to the characterization of endogenous forms of mature proteins from SDS-PAGE gels.

Animals↗
PubMed1997

Continuous venovenous haemofiltration using polyacrylonitrile filters does not activate contact system and intrinsic coagulation pathways.

OBJECTIVES: To investigate whether continuous venovenous haemofiltration using polyacrylonitrile filters causes activation of the contact system and intrinsic coagulation pathways and if this, and/or low plasma levels of endogenous anticoagulants, influences filter lifespan. DESIGN: Observational study. SETTING: University Teaching Hospital Intensive Care Unit. PATIENTS: Twelve critically ill patients with acute renal failure receiving continuous venovenous haemofiltration. INTERVENTIONS: Blood samples were taken before starting haemofiltration, at 15 min, 1 h, 3-4 h, 8-12 h, 24 h and at 24-h intervals thereafter until filter blockage occurred. Measurement was made of the contact and intrinsic coagulation system proteins factor XII, activated factor XII and prekallikrein and the protease inhibitors antithrombin III, heparin co-factor II, alpha 2-macroglobulin and C1-esterase inhibitor. Thrombin-antithrombin complex levels were measured to provide evidence of thrombin generation. RESULTS: (i) Factor XII, prekallikrein and contact system inhibitors were subnormal in 10/12 and activated factor XII raised in 11/12 patients at baseline, implying pre-existing contact pathway activation. (ii) No change occurred during haemofiltration in the intrinsic coagulation pathway factor or inhibitor levels. (iii) Clotting of the filter circuit within the first 24 h occurred in 5/12 and was associated with low baseline levels of antithrombin III and heparin co-factor II. Only in these patients did thrombin-antithrombin complex levels rise significantly. CONCLUSIONS: The contact system was not activated further by continuous venovenous haemofiltration using polyacrylonitrile filters in critically ill patients. Premature clotting of the haemofilter circuit was more common in patients with very low levels of antithrombin III and heparin co-factor II; although this was related to thrombin generation, the intrinsic coagulation pathway does not appear to be implicated.

Acrylic Resins↗
PubMed1997

Working with resistance to experiencing and expressing emotions in group therapy.

Emotional insight is essential in order for therapeutic change to take place. Profound resistances to experiencing, expressing, and understanding emotions are present in group members, the group as a whole, and the group therapist. Reasons for the three types of resistances are described. Specific therapist attitudes and therapeutic techniques are suggested to understand and effectively work through resistances at individual, interpersonal, and whole-group levels. Examples are given from an ongoing therapy group to illustrate the processes of understanding and working through resistances. Resistances on the part of the group therapist are identified and their possible effects on the group's resistance are explored.

Adult↗
PubMed1996

DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2-terminal domains stabilizes binding of two dimers to tandem DNA sites.

Stat1 alpha, Stat1 beta and a proteolytically defined truncated Stat1 (132-713, Stat1tc) have been prepared from recombinant sources. All three proteins were specifically phosphorylated on Tyr701 in vitro and the phosphoprotein purified to homogeneity. This was achieved by employing a new isolation scheme that does not include DNA affinity steps and readily allows for the isolation of tens of milligrams of activated Stat protein. The purified phosphoprotein was free of traces of unphosphorylated polypeptide as detected by mass spectrometry. The phosphorylated Stat1 preparations bound to various DNA recognition sites with the same Keq of approximately 1 x 10(-9) M; distinction between 'weak' and 'strong' binding sites is determined by the very rapid dissociation (< 30 s, t1/2) from 'weak' sites compared with 'strong' sites (approximately 3 min, t1/2). Reports of 'weak' tandem binding sites in a natural gene caused us to examine binding to tandem sites leading to the finding that the Stat1 alpha or beta (38 amino acids shorter on the C terminus) bound to two tandem sites (but not two head-to-head sites) with a higher stability than to a single recognition site. The N-terminally truncated protein Stat1tc did not show this cooperative binding, thus implicating the N-terminal domain in promoting Stat1-Stat1 dimer interaction.

Binding Sites↗
PubMed1996

Structural similarity between TAFs and the heterotetrameric core of the histone octamer.

A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution. The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix. Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure.

Amino Acid Sequence↗
PubMed1996

Influence of matrix solution conditions on the MALDI-MS analysis of peptides and proteins.

Sample-matrix preparation procedures are shown to greatly influence the quality of the matrix-assisted laser desorption/ionization (MALDI) mass spectra of peptides and proteins. In particular, dramatic mass discrimination effects are observed when the matrix 4-hydroxy-alpha-cyanocinnamic acid is used for analyzing complex mixtures of peptides and proteins. The discrimination effects are found to be strongly dependent on the sample-matrix solution composition, pH, and the rates at which the sample-matrix cocrystals are grown. These findings demonstrate the need to exercise great care in performing and interpreting the MALDI analysis of biological samples. The results also indicate that there is a reverse-phase chromatographic-like dimension in the sample-matrix preparation procedures that can be exploited to optimize the analysis. The present work describes the conditions under which the majority of components of a complex mixture of peptides and proteins can be successfully measured.

Amino Acid Sequence↗
PubMed1995

Weight-reducing effects of the plasma protein encoded by the obese gene.

The gene product of the ob locus is important in the regulation of body weight. The ob product was shown to be present as a 16-kilodalton protein in mouse and human plasma but was undetectable in plasma from C57BL/6J ob/ob mice. Plasma levels of this protein were increased in diabetic (db) mice, a mutant thought to be resistant to the effects of ob. Daily intraperitoneal injections of either mouse or human recombinant OB protein reduced the body weight of ob/ob mice by 30 percent after 2 weeks of treatment with no apparent toxicity but had no effect on db/db mice. The protein reduced food intake and increased energy expenditure in ob/ob mice. Injections of wild-type mice twice daily with the mouse protein resulted in a sustained 12 percent weight loss, decreased food intake, and a reduction of body fat from 12.2 to 0.7 percent. These data suggest that the OB protein serves an endocrine function to regulate body fat stores.

Adipose Tissue↗