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S Lee

Publications and source records attributed to S Lee.

At least 829 records · Page 46Linked to original sources
PubMed1996

Design and synthesis of amphiphilic alpha-helical model peptides with systematically varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membranes.

Five amphiphilic alpha-helical peptides of 18 residues containing a hydrophobic Trp residue as a fluorescence probe were designed. The peptides were made up of hydrophobic Leu and hydrophilic Lys residues of a ratio of 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively). These peptides generate ideal amphiphilic alpha-helical structures, which have systematically varied hydrophobic-hydrophilic balance (relative amphiphilic potential) as a result of different hydrophobicities and almost the same hydrophobic moments. Their hydrophobic-hydrophilic balance was estimated both theoretically from the calculated hydrophobicity values (or the magnitude of hydrophobic faces) and experimentally from the retention times in reverse phase high-performance liquid chromatography (RP-HPLC). Circular dichroism, liposome-lytic, and Trp-fluorescent studies in buffer and in the presence of acidic and neutral liposomes clearly showed that the increasing hydrophobic face area not only increases the affinity for lipid but also increases the trend of self-association. The structure-activity relationship estimated by means of leakage ability and hemolytic activity demonstrated that the model- and bio-membrane perturbation ability is completely parallel to the magnitude of the hydrophobic face area. The lipid-binding study in guanidine hydrochloride solution showed that the peptides with a hydrophobic face larger than the hydrophilic face (Hels 13-5 and 11-7) immerse their hydrophobic regions in lipid bilayers and that the inverse ones (Hels 7-11 and 5-13) interact only between the anionic lipid head groups and cationic peptide residues on liposome surfaces. The peptide Hel 9-9, which has exactly the same hydrophobic and hydrophilic regions, was found to be at a critical boundary among these peptides in terms of (1) behavior of peptide self-aggregation in buffer solution and membrane perturbation ability, (2) transfer from bulk solution to neutral lipid bilayers, and (3) necessity of charge interaction in lipid-peptide binding.

Amino Acid Sequence↗
PubMed1996

Structural analysis of Sindbis virus capsid mutants involving assembly and catalysis.

Sindbis virus core protein (SCP) has been isolated from virus and crystallized. The X-ray crystallographic structure showed that the amino-terminal 113 residues appeared to be either disordered or truncated during crystallization and that the carboxy-terminal residues 114 to 264 had a chymotrypsin-like structure. The carboxy-terminal residues 106 to 264 and 106 to 266 of SCP have now been expressed in Escherichia coli. Most crystal forms of the truncated proteins were isomorphous with those of the virally extracted protein. There are only small structural differences between the truncated recombinant protein and the ordered part of the wild-type virus-extracted protein. Hence, E. coli-expressed SCP can be used to study proteolytic properties and the contribution of SCP to nucleocapsid assembly, interaction with the E2 glycoprotein and interaction with RNA. The same dimer that was found in two different crystal forms of the virus-extracted SCP was present also in some of the crystals of the truncated recombinant protein. The monomer-monomer interface is maintained by two pairs of hydrogen bonds and by hydrophobic interactions. Removal of the hydrogen bonds by single substitutions did not prevent dimer formation. However, a mutation that reduced the hydrophobic contacts did inhibit dimer formation. The wild-type truncated SCP is active in E. coli, as evidenced by proteolytic processing of a series of progressively longer precursors that extend beyond residue 264. Unlike the virus-extracted capsid protein, the E. coli-expressed SCP described here is terminated following the carboxy-terminal residue and, therefore, does not require autocatalysis. Nevertheless, the E. coli-expressed protein folds with the carboxy-terminal tryptophan residue in the specificity pocket. Two crystallographically independent molecules of SCP(106 to 266), which had two additional downstream residues and had the essential S215 mutated to alanine, showed two distinct modes of binding the uncleaved carboxy-terminal residues. These may represent successive steps of binding substrate prior to catalytic cleavage. Refinement of the various crystal structures of SCP showed that the amino-terminal arm from residues 107 to 113 was not disordered, but is associated with neighboring molecules. Residues 108 to 111 bind into a hydrophobic pocket composed primarily of Y180, W247 and F166. It had been shown that the double mutant (Y180S; E183G), with the Y180S substitution in this pocket, produced a large number of non-infectious virions, possibly because of modification in the interaction of the glycoprotein spikes with core proteins. The crystal structure of this double mutant showed that there was a large positional change in the side-chain of W247, which moved into the space created by the replacement of Y180 with serine. These conformational changes may alter the stability of the virion and, thus, regulate its functional requirements during cell entry.

3C Viral Proteases↗
PubMed1996

Long-term modifications of synaptic efficacy in the human inferior and middle temporal cortex.

The primate temporal cortex has been demonstrated to play an important role in visual memory and pattern recognition. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here we address this issue by examining the induction of synaptic plasticity in surgically resected human inferior and middle temporal cortex. The results show that synaptic strength in the human temporal cortex could undergo bidirectional modifications, depending on the pattern of conditioning stimulation. High frequency stimulation (100 or 40 Hz) in layer IV induced long-term potentiation (LTP) of both intracellular excitatory postsynaptic potentials and evoked field potentials in layers II/III. The LTP induced by 100 Hz tetanus was blocked by 50-100 microM DL-2-amino-5-phosphonovaleric acid, suggesting that N-methyl-D-aspartate receptors were responsible for its induction. Long-term depression (LTD) was elicited by prolonged low frequency stimulation (1 Hz, 15 min). It was reduced, but not completely blocked, by DL-2-amino-5-phosphonovaleric acid, implying that some other mechanisms in addition to N-methyl-DL-aspartate receptors were involved in LTD induction. LTD was input-specific, i.e., low frequency stimulation of one pathway produced LTD of synaptic transmission in that pathway only. Finally, the LTP and LTD could reverse each other, suggesting that they can act cooperatively to modify the functional state of cortical network. These results suggest that LTP and LTD are possible mechanisms for the visual memory and pattern recognition functions performed in the human temporal cortex.

2-Amino-5-phosphonovalerate↗
PubMed1996

Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin.

Alcohol (EtOH) treatment increases plasma ACTH levels in the rat, a response thought to depend at least partially on endogenous corticotropin-releasing factor (CRF). However, the inability of CRF antibodies to completely abolish EtOH-induced activation of the pituitary has suggested the possible importance of other factors. Because vasopressin (VP) may be acutely released by alcohol in the rat, and because this peptide interacts with CRF to promote ACTH secretion, we investigated its role in the hypothalamic-pituitary (HP) axis' response to one acute injection of EtOH (3 g/kg, i.p.). As expected, blockade of pituitary CRF receptors significantly blunted EtOH-induced ACTH secretion. Administration of a VP antagonist also interfered with this response, though to a lesser extent, while concomitant administration of both CRF and VP antagonists totally abolished it. These results indicate that both CRF and VP participate in the ability of alcohol to stimulate corticotrophs' function. Another hypothesis still awaiting confirmation is whether alcohol stimulates the activity of hypothalamic neurons important for ACTH release, which are primarily found in the paraventricular nucleus (PVN) of the hypothalamus. We therefore determined whether EtOH upregulated transcripts of the immediate early gene (IEG) NGFI-B, as well as those of CRF and VP. In the parvicellular portion of the PVN (pPVN), NGFI-B mRNA and CRF heteronuclear (hn) RNA levels increased within 20 min of EtOH treatment. Despite this robust response, densitometric assessments of CRF mRNA levels failed to indicate a significant elevation over control levels at any of the time points studied, a phenomenon that might be at least in part due to the abundance of these transcripts under basal conditions. Constitutive levels of VP hnRNA levels were very low in the pPVN, but high in the magnocellular division (mPVN). These signals tended to be increased in rats injected with alcohol, but these responses did not reach statistical significance. However, the mRNA encoding VP became significantly more expressed 3 h post-alcohol exposure, suggesting that gene expression had increased. Collectively, these results indicate that alcohol stimulates the activity of neurons in both the parvo- and the magnocellular portions of the PVN, though transcriptional activation of the CRF and VP genes in the PVN exhibit distinct time courses. Whether the influence of the drug is direct or is exerted through afferents to the PVN, and whether increased hypothalamic neuronal activity plays a role in mediating the acute ACTH response to alcohol, are issues that have not yet been adequately resolved.

Adrenocorticotropic Hormone↗
PubMed1996

Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB.

The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B". Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides. The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences. The gene encoding TFIIIB90 is essential for viability of yeast. Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1. In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex. To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA. Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB.

Amino Acid Sequence↗
PubMed1996

Quality assurance issues in DNA image cytometry.

DNA image cytometry is currently used in many clinical laboratories as a prognostic tool for the study of patients with malignant neoplasms. However, quality assurance (QA) procedures for image cytometry (IC) have not been standardized. National proficiency testing programs for DNA IC are yet to be developed. We describe our QA program for DNA IC. Indicators of quality; thresholds for evaluation; methods for collection, organization, and evaluation of data; and actions recommended to deal with problems are described for each of the phases of DNA IC. They include QA procedures for sample acceptability; staining technique; instrumentation, including linearity and calibration verification; and correlation with other cytometric methods. The difficult problem of standardizing the postanalytical phase of QA is discussed. The need for a consensus conference to adopt standardized QA procedures for DNA image analysis is emphasized.

DNA, Neoplasm↗
PubMed1996

A quasi-monoclonal mouse.

As a model for studying the generation of antibody diversity, a gene-targeted mouse was produced that is hemizygous for a rearranged V(D)J segment at the immunoglobulin (Ig) heavy chain locus, the other allele being nonfunctional. The mouse also has no functional kappa light chain allele. The heavy chain, when paired with any lambda light chain, is specific for the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP). The primary repertoire of this quasi-monoclonal mouse is monospecific, but somatic hypermutation and secondary rearrangements change the specificity of 20 percent of the antigen receptors on B cells. The serum concentrations of the Ig isotypes are similar to those in nontransgenic littermates, but less than half of the serum IgM binds to NP, and none of the other isotypes do. Thus, neither network interactions nor random activation of a small fraction of the B cell population can account for serum Ig concentrations.

Animals↗
PubMed1996

Importance of pituitary and brain receptors for corticotrophin-releasing factor in modulating alcohol-induced ACTH secretion in the rat.

Alcohol (EtOH) releases ACTH through mechanisms that ultimately depend on the presence of endogenous corticotropin-releasing factor (CRF), but we still do not know where alcohol acts within the hypothalamic-pituitary-adrenal (HPA) axis. The present study was designed to determine the respective importance of the activation of pituitary and/or hypothalamic CRF receptors in mediating the stimulatory effect of EtOH on ACTH secretion. We used two CRF antagonists: (1) alpha-helical CRF9-41 (alpha-hel ant.), which is very effective in interfering with biological responses mediated by brain CRF receptors, but relatively impotent in blocking pituitary CRF receptors; and (2) astressin, {cyclo(30-33)[DPhe12,Nle21,38,Glu30,Lys33]r/h CRF12.41}, a member of the newest generation of antagonists capable of significantly blocking both brain and pituitary CRF receptors. The role of pituitary CRF receptors was shown by the ability of i.v. injected astressin, at doses shown to completely block CRF-induced ACTH release, to significantly (P < 0.01) reduce the ACTH response to EtOH (1.5 or 3 g/kg, i.p.). The importance of hypothalamic CRF receptors was tested by injecting alpha-hel ant. intracerebroventricularly (i.c.v.). We reasoned that if this antagonist leaked to the pituitary following administration into the ventricle, it would not be able to act directly on the corticotrophs. The ACTH response to EtOH was modestly and not significantly (P > 0.05) blunted by i.c.v. alpha-hel ant. Similarly, the stimulatory effect of alcohol on hypothalamic neuronal activation, measured by increases in the immediate early gene NGFI-B mRNA levels, was only slightly altered by blockade of hypothalamic CRF receptors. Collectively, these results suggest that during acute alcohol treatment, few brain pathways mediating the stimulatory effect of EtOH on ACTH release depend on the activation of CRF synapses. The induction of immediate early gene transcripts by alcohol similarly relies on hypothalamic circuits that stimulate CRF neurons relatively independently of the activation of CRF receptors. In contrast, functional pituitary CRF receptors are essential for the ACTH response to the drug.

Adrenocorticotropic Hormone↗
PubMed1996

Identification of a protein binding site on the surface of the alphavirus nucleocapsid and its implication in virus assembly.

BACKGROUND: Many enveloped viruses exit cells by budding from the plasma membrane. The driving force for budding is the interaction of an inner protein nucleocapsid core with transmembrane glycoprotein spikes. The molecular details of this process are ill defined. Alphaviruses, such as Sindbis virus (SINV) and Semliki Forest virus (SFV), represent some of the simplest enveloped viruses and have been well characterized by structural, genetic and biochemical techniques. Although a high-resolution structure of an alphavirus has not yet been attained, cryo-electron microscopy (cryo-EM) has been used to show the multilayer organization at 25 A resolution. In addition, atomic resolution studies are available of the C-terminal domain of the nucleocapsid protein and this has been modeled into the cryo-EM density. RESULTS: A recombinant form of Sindbis virus core protein (SCP) was crystallized and found to diffract much better than protein extracted from the virus (2.0 A versus 3.0 A resolution). The new structure showed that amino acids 108 to 111 bind to a specific hydrophobic pocket in neighboring molecules. Re-examination of the structures derived from virus-extracted protein also showed this 'N-terminal arm' binding to the same hydrophobic pocked in adjacent molecules. It is proposed that the binding of these capsid residues into the hydrophobic pocket of SCP mimics the binding of E2 (one of two glycoproteins that penetrate the lipid bilayer of the viral envelope) C-terminal residues in the pocket. Mutational studies of capsid residues 108 and 110 confirm their role in capsid assembly. CONCLUSIONS: Structural and mutational analyses of residues within the hydrophobic pocket suggest that budding results in a switch between two conformations of the capsid hydrophobic pocket. This is the first description of a viral budding mechanism in molecular detail.

Alphavirus↗
PubMed1996

Crystal structure of the yeast TFIIA/TBP/DNA complex.

The crystal structure of the yeast TFIIA/TBP/TATA promoter complex was solved to 3 angstrom resolution by double-edge multiple wavelength anomalous diffraction from two different species of anomalous scattering elements in the same crystal. The large and small subunits of TFIIA associate intimately to form both domains of a two-domain folding pattern. TFIIA binds as a heterodimer to the side of the TBP/TATA complex opposite to the side that binds TFIIB and does not alter the TBP/DNA interaction. The six-stranded beta-sandwich domain interacts with the amino-terminal end of TBP through a stereospecific parallel beta-strand interface and with the backbone of the TATA box and the 5'-flanking B-DNA segment. The four-helix-bundle domain projects away from the TBP/TATA complex, thereby presenting a substantial surface for further protein-protein interactions.

Amino Acid Sequence↗
PubMed1996

CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response.

The ligand for CD40 (CD40L) is expressed on the surface of activated CD4+ T cells and its role in T-B cell collaborations and thymus-dependent humoral immunity is well established. Recently, by generating CD40L-knockout mice, we have confirmed its previously described role in humoral immunity and defined another important function of this molecule in the in vivo clonal expansion of antigen-specific CD4+ T cells. Here, we investigated the potential in vivo role of CD40L in antiviral immunity by examining the immune response mounted by CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV), Pichinde virus, or vesicular stomatitis virus. Humoral immune responses of CD40L-deficient mice to these viruses were severely compromised, although moderate titres of antiviral IgM and some IgG2a were produced by virus-infected CD40L-deficient mice by a CD4+ T cell-independent mechanism. By contrast, CD40L-deficient mice made strong primary CTL responses to all three viruses. Interestingly however, although memory CTL activity was detectable in CD40L-deficient mice two months after infection with LCMV, the memory CTL response was much less efficient than in wild-type mice. Together, the results show that CD40-CD40L interactions are required for strong antiviral humoral immune responses, and reveal a novel role for CD40L in the establishment and/or maintenance of CD8+ CTL memory.

Animals↗
PubMed1996

Homogeneous NMR spectra in inhomogeneous fields.

Researchers interested in high-resolution nuclear magnetic resonance (NMR) spectroscopy have long sought higher magnetic fields to enhance resolution and simplify spectra. Magnets with substantially larger fields than those available in the best commercial spectrometers are available, but the inhomogeneity is unacceptable for high-resolution spectra. A detection method (termed HOMOGENIZED) is presented that removes inhomogeneity while retaining chemical shift differences and J couplings. With existing inhomogeneous magnets, this method could nearly double the largest resonance frequency available for high-resolution NMR. The HOMOGENIZED sequence is based on observations of intermolecular zero-quantum coherences between a solute molecule and solvent molecules that are micrometers away; as long as the field is homogeneous over this short distance, sharp resonances are recovered without echoes. Experimental demonstrations and a detailed density matrix theory to explain the effect are presented.

Aprotinin↗