Biomedical subjects

S Lin

Publications and source records attributed to S Lin.

At least 307 records · Page 17Linked to original sources

PubMed1998

Detection of non-covalent interaction of single and double stranded DNA with peptides by MALDI-TOF.

DNA-histone interaction facilitates packaging of huge amounts of DNA in the confined space of the nucleus. The importance of this interaction underscores the need for new analytical techniques to acquire a better understanding of nuclear dynamics. Electrospray-ionization mass spectrometry made it possible to investigate non-covalently-bound biopolymers. We are enlarging the scope of available analytical tools by studying non-covalent interaction between single and double stranded DNA and peptides with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The interaction is an ionic one, between the negatively charged sugar-phosphate backbone of single stranded DNA and positively charged side chains of Arg- and Lys-rich peptides as demonstrated by Vertes' group with the dipeptides Arg-Lys and His-His. We replicated Lecchi and Pannell's work, which showed that double stranded DNA could be seen by MALDI using 6-aza-2-thiothymine (ATT) as matrix. We tried various peptides and found that as was demonstrated in DNA-histone interaction, a certain ratio and arrangement of basic residues was needed in order to generate ionic binding between DNA and peptide. We tested various single and double stranded DNA with the peptide of choice, and found that other variables such as pH value of solution, ionic strength, and matrix system did play a role.

DNA↗

PubMed1997

Preliminary results from intensity-based CT-SPECT fusion in I-131 anti-B1 monoclonal-antibody therapy of lymphoma.

BACKGROUND: In treatment of non-Hodgkin's lymphoma patients with predose-plus-I-131-labeled anti-B1 (anti-CD20) monoclonal antibody, an intratherapy single photon emission computed tomography (SPECT) image is an important part of research estimates of tumor dosimetry. For that imaging, a computed tomography (CT)-SPECT fusion is used both to obtain an attenuation map for the space-alternating generalized expectation maximization reconstruction and to provide CT-based volumes of interest (VoI) to determine activity in tumors and organs. Fusion based on external, skin-surface markers has been used but may not correctly superimpose internal structures. METHODS: A new algorithm, developed and implemented in the Department of Radiology, University of Michigan, and based on the mutual information of grayscale values, was investigated. Results from four anti-B1 therapy patients are presented. RESULTS: In one patient, the new intensity-based fusion provided total reconstructed counts for kidneys that were higher than those produced by marker-based fusion; therefore, the VoI was probably located more accurately. In a second patient, after an acquisition that did not include any skin markers, the new algorithm produced counts/pixel that were similar for four of five tumors consistent with what is expected from an ideal therapy combined with accurate count density estimates. The fifth tumor was quite small and will have its final activity estimate moved toward consistency with the others after a recovery coefficient multiplication. For four tumors in two patients, direct comparison of the two algorithms yielded count totals that were different by no more than 7.2%. CONCLUSIONS: The use of CT-SPECT fusion and subsequent transfer of tumor VoI originally drawn in high-resolution CT space offers potential advantages for quantifying tumor uptake of radioactivity. A new, mutual-information-based fusion algorithm is usable without skin markers. Results indicate that the new fusion algorithm gives equal tumor count values within 7.2% compared with fusion based on external markers. It increases estimates of kidney activity by an average of 6.4%.

Algorithms↗

PubMed1997

Determination of the dissociation constant of phosvitin-anti-phosphoserine interaction by affinity capillary electrophoresis.

We used affinity capillary electrophoresis (ACE) to study the interaction of a monoclonal anti-phosphoserine antibody (mAb) to a homopolyvalent antigen (hpAg), phosvitin. A model system, which allows the measurement of the true dissociation constant (Kd) in Ag excess based on measurement of migration shifts of mAb-hpAg complexes at different Ag concentrations in solution, is presented for the study of the interactions between a mAb and an Ag that has identical determinants. The experimental value of Kd (22.4 x 10(-6) M) obtained by ACE is shown to be in close agreement with the value (17.8 x 10(-6) M) obtained by the conventional immunoassay based on indirect competition enzyme-linked immunosorbent assay (ELISA). Moreover, the Kds of mAb-hpAg complexes were measured and shown to be independent of the applied electrical field strength. Thus, under conditions where the total Ag concentration is in large excess over the total Ab concentration and when certain requirements are fulfilled, this method offers the advantage of dealing with the determination of Kd for unlabeled mAb and homopolymeric Ag molecules in free solution rather than at the liquid-solid interface.

Antibodies, Monoclonal↗

PubMed1997

Site-specific fluorescence labeling of the beta2 adrenergic receptor amino terminus.

A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.

Adenylyl Cyclases↗

PubMed1997

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

Adrenergic beta-Agonists↗

PubMed1997

Fasting increases leptin receptor mRNA expression in lean but not obese (ob/ob) mouse brain.

Using a non-radioactive in situ hybridization detection method, we found that fasting up-regulated leptin receptor mRNA expression in the brain of C57 Bl/6 lean (+/+) but not obese (ob/ob) mice. After the lean mice were fasted for 24 h, increased leptin receptor mRNA expression was found in the arcuate and ventromedial hypothalamic nuclei, medial habenular nucleus and dentate gyrus of hippocampal formation. There was only a minor increase in mRNA expression of leptin receptor in the hippocampus proper while the piriform cortex remained unchanged. However, after the obese mice were fasted for 24 h, no detectable changes of leptin receptor mRNA expression could be found. These results indicated that leptin receptor gene expression was influenced by nutritional status. Further, genetically obese mice showed an inability to respond to nutritional change, probably due to the fact that obese mice lack leptin.

Animals↗

PubMed1997

Tyrosine-dependent basolateral sorting signals are distinct from tyrosine-dependent internalization signals.

Converting cysteine 543 to tyrosine in the influenza virus hemagglutinin (HA) introduces both a basolateral sorting signal and an internalization signal into the HA cytoplasmic domain. Another HA mutant, HA+8, contains eight additional amino acids at the end of the cytoplasmic domain that include a powerful internalization signal. HA+8 was also sorted efficiently to the basolateral surface of Madin-Darby canine kidney cells. The simplest explanation for the observation that multiple sorting phenotypes depend upon the same small amino acid sequence is that certain tyrosine-based internalization signals might also function as basolateral sorting signals. To test this hypothesis, second-site mutations were introduced into HA C543Y or HA+8 to determine if the internalization and basolateral sorting functions can be separated. For HA C543Y, the same sequence positions were important for both basolateral sorting and internalization, but the two functions responded differently to individual amino acid replacements, indicating that they were distinct. For HA+8, the basolateral sorting signal required the same tyrosine as the internalization signal, but did not share any other characteristics. Thus, even when basolateral sorting signals that depend on tyrosine overlap or are co-linear with internalizations signals, the two sorting processes are sensitive to different characteristics of the sequence.

Amino Acid Sequence↗

PubMed1997

Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.

We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping layer (EVL), and neuronal expression of GATA-2. During gastrulation, GFP expression is confined to the ventral ectoderm and lateral mesoderm and is lacking in the dorsal shield. Cells derived from the regions expressing GFP give rise to hematopoietic progenitors, EVL cells, and neurons. Deletion analysis of the 7.3-kb GATA-2 promoter region revealed that a 1.1-kb DNA sequence is critical for expression of GATA-2 in neurons. Fine mapping revealed that a 31-bp region is required for neuron enhancer activity, and mutagenesis showed that the DNA motif CCCTCCT is essential for GATA-2 promoter activity in the central nervous system of zebrafish. Our use of zebrafish embryos can be exploited as a whole animal system for the dissection of any developmentally regulated vertebrate promoter.

Animals↗

PubMed1997

Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.

The voltage activated K+ channel (Kv1.3) has recently been identified as the molecule that sets the resting membrane potential of peripheral human T lymphoid cells. In vitro studies indicate that blockage of Kv1.3 inhibits T cell activation, suggesting that Kv1.3 may be a target for immunosuppression. However, despite the in vitro evidence, there has been no in vivo demonstration that blockade of Kv1.3 will attenuate an immune response. The difficulty is due to species differences, as the channel does not set the membrane potential in rodent peripheral T cells. In this study, we show that the channel is present on peripheral T cells of miniswine. Using the peptidyl Kv1.3 inhibitor, margatoxin, we demonstrate that Kv1.3 also regulates the resting membrane potential, and that blockade of Kv1.3 inhibits, in vivo, both a delayed-type hypersensitivity reaction and an Ab response to an allogeneic challenge. In addition, prolonged Kv1.3 blockade causes reduced thymic cellularity and inhibits the thymic development of T cell subsets. These results provide in vivo evidence that Kv1.3 is a novel target for immunomodulation.

Animals↗

PubMed1997

Time-resolved spectroscopy of energy transfer and trapping upon selective excitation in membranes of Heliobacillus mobilis at low temperature.

Transient absorption difference spectra in the Qy absorption band of bacteriochlorophyll (BChl) g and in the 670 nm absorption band of the primary acceptor A0 in membranes of Heliobacillus mobilis (Hc. mobilis) were measured at 20 K upon selective excitation at 668, 793, 810, and 815 nm with a 5 nm spectral bandwidth. When excited at 793 nm, the spectral equilibration of excitations from shorter to longer wavelength-absorbing pigments occurred within 3 ps and mostly localized at the band centered around 808 nm. When excited at 668 nm, the excitation energy transfer from the 670 nm absorbing pigment to the Qy band of BChl g took less than 0.5 ps, and the energy redistribution occurred and localized at 808 nm as in the case of the 793 nm excitation. All of the excitations were localized at the long wavelength pigment pool centered around 810 or 813 nm when excited at 810 or 815 nm. A slower energy transfer process with a time constant of 15 ps was also observed within the pool of long wavelength-absorbing pigments upon selective excitation at different wavelengths as has been observed by Lin et al. (Biophys. J. 1994, 67, 2479) when excited at 590 nm. Energy transfer from long wavelength antenna molecules to the primary electron donor P798 followed by the formation of P+ took place with a time constant of 55-70 ps for all excitations. Direct excitation of the primary electron acceptor A0, which absorbed at 670 nm, showed the same kinetic behavior as in the case when different forms of antenna pigments were excited in the Qy region. This observation generally supports the trapping-limited case of energy transfer in which the excitations have high escape probability from the reaction center (RC) until the charge separation takes place. Possible mechanisms to account for the apparent "uphill" energy transfer from the long wavelength antenna pigments to P798 are discussed.

Bacteria↗

PubMed1997

Syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) and analogs with 13C- and 2H-labeled choline head groups.

The syntheses of four head group labeled analogs of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) (6) by a general method from 1,2-di-O-palmitoyl-sn-glycero-3-phosphatidic acid (5) have been performed. The syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phospho[alpha-13C]choline (6a) and 1,2-di-O-palmitoyl-sn-glycero-3-phospho[beta-13C]choline (6b) were performed from labeled [1-13C]glycine (1a) in 52% overall yield and from [2-13C]glycine (1b) in 56% overall yield, respectively. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[N(C2H3)3]choline (9) was prepared from 2-aminoethanol in 39% overall yield. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[alpha-C2H2]choline (12) was prepared from N,N-dimethylglycine ethyl ester in 50% overall yield.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Self-organization of firing activities in monkey's motor cortex: trajectory computation from spike signals.

The population vector method has been developed to combine the simultaneous direction-related activities of a population of motor cortical neurons to predict the trajectory of the arm movement. In this article, we consider a self-organizing model of a neural representation of the arm trajectory based on neuronal discharge rates. As self-organizing feature map (SOFM) is used to select the optimal set of weights in the model to determine the contribution of an individual neuron to an overall movement representation. The correspondence between movement directions and discharge patterns of the motor cortical neurons is established in the output map. The topology-preserving property of the SOFM is used to analyze the recorded data of a behaving monkey. The data used in this analysis were taken while the monkey was tracing spirals and doing center-->out movements. The arm trajectory could be well predicted using such a statistical model based on the motor cortex neuronal firing information. The SOFM method is compared with the population vector method, which extracts information related to trajectory by assuming that each cell has a fixed preferred direction during the task. This implies that these cells are acting along lines labeled only for direction. However, extradirectional information is carried in these cell responses. The SOFM has the capability of extracting not only direction-related information but also other parameters that are consistently represented in the activity of the recorded population of cells.

Algorithms↗

PubMed1997

Specific mutation near the primary donor in photosystem I from Chlamydomonas reinhardtii alters the trapping time and spectroscopic properties of P700.

Time-resolved absorption and fluorescence spectroscopy were used to investigate the energy and electron transfer processes in the detergent-isolated photosystem I core particles from the site-directed mutant of Chlamydomonas reinhardtii with the histidine-656 of PsaB replaced by asparagine [HN(B656) mutation]. The specific mutation near the primary donor molecule results in a 40 mV increase in the P700/P700+ midpoint potential [Webber, A. N., Su Hui, Bingham, S. E., Kass, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., & Lubitz, W. (1996) Biochemistry 35, 12857-12863]. There is no indication that the HN(B656) mutation affects the spectral distribution of the antenna pigments. However, the lifetime of the trapping process measured independently by transient absorption and fluorescence spectroscopy in the mutant PSI core antenna is increased by a factor of approximately 2 (approximately 65 ps compared to approximately 30 ps in the wild-type PSI). This implies that the trapping process in the PSI antenna is limited by the process where the primary donor molecule directly participates. The HN(B656) mutation results in the appearance of a new bleaching band at 670 nm in the spectrum which is due to formation of P700+ upon photooxidation. The difference spectrum of the photoreduction of the possible primary acceptor, A0 in the mutant PSI is very similar to wild type, indicating that it is unaffected by the HN(B656) mutation. Possible mechanisms for slowing of the trapping process and the appearance of a new band in the P700 - P700+ difference spectrum of the HN(B656) PSI are discussed.

Animals↗

PubMed1997

Upregulation of leptin receptor mRNA expression in obese mouse brain.

Leptin receptor gene expression in the brains of lean (+/+) and obese (ob/ob) C57Bl/6 mice was examined using a non-radioactive in situ hybridization detection method. Significant increases in leptin receptor mRNA expression were found in the ventromedial and arcuate hypothalamic nuclei, piriform and olfactory cortices and medial habenular nucleus. There were very minor changes in the amount of leptin receptor mRNA expression in hippocampus proper (CA1-3). Results indicated that leptin receptor is upregulated when there is a lack of functional leptin, as in hereditary obese (ob/ob) mice. It is also suggested that leptin receptor may be an autoreceptor.

Animals↗

PubMed1997

Effect of glucose concentration on swimming motility in enterobacteria.

Since the observation that glucose prevents the synthesis of flagella in Escherichia coli was first reported in 1967, many studies have addressed the underlying mechanism. Currently, it is thought that an increase in glucose concentration decreases the intracellular CRP/cAMP concentration. This leads to an inhibitory effect on the expression of the flhD operon, the master operon for flagella synthesis. In our study on defining factors influencing the cell differentiation of Serratia marcescens, glucose catabolite repression of hag expression and swimming/swarming motility was not observed. Further experiments using a simple swimming motility assay extended this observation to other members of Enterobacteriaceae. Although the underlying mechanism is still uncharacterised, our results suggest that glucose catabolite repression of swimming motility may not be a common phenomenon in Enterobacteriaceae.

DNA-Binding Proteins↗

PubMed1997

Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of gamma-glutamyl hydrolase activity.

A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates2 using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme gamma-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu4) is achieved within 10 min using a 75 microns I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5 x 10(5) eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.

Electrophoresis, Capillary↗

PubMed1997

Identification and characterization of a predominant isoform of human MKK3.

We have obtained a novel cDNA species of MKK3, termed MKK3b. MKK3b cDNA differs from its original form, MKK3, at the 5'-end, encoding 29 extra amino acids in the N-terminus. Analysis of MKK3 genomic DNA structure revealed that the MKK3b-unique 5'-end sequence is derived from an exon different from that of MKK3, and that they share identical sequences thereafter. This suggests that the two cDNA forms of MKK3 are either generated by differential splicing of the same gene or derived from differential promoter utilization. Northern blotting analysis showed that MKK3b mRNA is much more abundant than MKK3. Functional characterization based on the activation of p38 revealed that MKK3b is more efficient than MKK3 in mediating downstream signalling events.

Amino Acid Sequence↗

PubMed1997

Fatty acid sulfonyl fluorides inhibit anandamide metabolism and bind to the cannabinoid receptor.

Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (palmitylsulfonyl fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50 values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylfluoride had an IC50 of 18 nM and C20 sulfonylfluoride had an IC50 of 78 microM. The C14, C16, and C18 sulfonyl fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.

Amidohydrolases↗