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Biomedical subjects

S Lin

Publications and source records attributed to S Lin.

At least 91 records · Page 5Linked to original sources
PubMed2001

Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys.

Collectrin, a novel homolog of angiotensin-converting enzyme-related carboxypeptidase (ACE2), was identified during polymerase chain reaction-based cDNA subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic phase. Collectrin, with 222 amino acids, has an apparent signal peptide and a transmembrane domain; the sequence is conserved in mouse, rat, and human and shares 81.9% identity. Human collectrin has 47.8% identity with non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin lacks active dipeptidyl carboxypeptidase catalytic domains. The collectrin mRNA transcripts are expressed exclusively in the kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows that it is localized to the luminal surface and cytoplasm of collecting ducts. Immunoprecipitation studies, using [35S]methionine-labeled renal cortical and inner medullar collecting duct cells, i.e. M-1 and mIMCD-3, indicate that the protein size is approximately 32 kDa. During the development of mouse kidney, mRNA signal is detectable at day 13 of gestation, and the protein product is observed in the ureteric bud branches. Its expression is progressively increased during later stages of the gestation extending into the neonatal periods and then is decreased in adult life. Up-regulated expression of collectrin in the hypertrophic kidneys after renal ablation and restricted spatio-temporal expression during development indicates a possible role(s)in the process of progressive renal failure and renal organogenesis.

Amino Acid Sequence↗
PubMed2001

Statistical optimization of gastric floating system for oral controlled delivery of calcium.

The development of an optimized gastric floating drug delivery system is described. Statistical experimental design and data analysis using response surface methodology is also illustrated. A central, composite Box-Wilson design for the controlled release of calcium was used with 3 formulation variables: X1 (hydroxypropyl methylcellulose [HPMC] loading), X2 (citric acid loading), and X3 (magnesium stearate loading). Twenty formulations were prepared, and dissolution studies and floating kinetics were performed on these formulations. The dissolution data obtained were then fitted to the Power Law, and floating profiles were analyzed. Diffusion exponents obtained by Power Law were used as targeted response variables, and the constraints were placed on other response variables. All 3 formulation variables were found to be significant for the release properties (P <.05), while only HPMC loading was found to be significant for floating properties. Optimization of the formulations was achieved by applying the constrained optimization. The optimized formulation delivered calcium at the release rate of 40 mg/hr, with predicted n and T50% values at 0.93 and 3.29 hours, respectively. Experimentally, calcium was observed to release from the optimized formulation with n and T50% values of 0.89 (+/- 0.10) and 3.20 (+/- 0.21) hours, which showed an excellent agreement. The quadratic mathematical model developed could be used to further predict formulations with desirable release and floating properties.

Administration, Oral↗
PubMed2001

Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.

Certain strains of Escherichia coli and Salmonella contain lipopolysaccharide (LPS) modified with a phosphoethanolamine (pEtN) group at position 7 of the outer 3-deoxy-d-manno-octulosonic acid (Kdo) residue. Using the heptose-deficient E. coli mutant WBB06 (Brabetz, W., Muller-Loennies, S., Holst, O., and Brade, H. (1997) Eur. J. Biochem. 247, 716-724), we now demonstrate that the critical parameter determining the presence or absence of pEtN is the concentration of CaCl(2) in the medium. As judged by mass spectrometry, half the LPS in WBB06, grown on nutrient broth with 5 mm CaCl(2), is derivatized with a pEtN group, whereas LPS from WBB06 grown without supplemental CaCl(2) is not. Membranes from E. coli WBB06 or wild-type W3110 grown on 5-50 mm CaCl(2) contain a novel pEtN transferase that uses the precursor Kdo(2)-[4'-(32)P]lipid IV(A) as an acceptor. Transferase is not present in membranes of E. coli grown with 5 mm MgCl(2), BaCl(2), or ZnCl(2). Hydrolysis of the in vitro reaction product, pEtN-Kdo(2)-[4'-(32)P]lipid IV(A), at pH 4.5 shows that the pEtN substituent is located on the outer Kdo moiety. Membranes from an E. coli pss knockout mutant grown on 50 mm CaCl(2), which lack phosphatidylethanolamine, do not contain measurable transferase activity unless exogenous phosphatidylethanolamine is added back to the assay system. The induction of the pEtN transferase by 5-50 mm CaCl(2) suggests possible role(s) in establishing transformation competence or resisting environmental stress, and represents the first example of a regulated covalent modification of the inner core of E. coli LPS.

Calcium↗
PubMed2001

Limited acid hydrolysis as a means of fragmenting proteins isolated upon ProteinChip array surfaces.

ProteinChip array technology enables protein purification, protein profiling, and biomarker discovery on a convenient biochip platform. Traditional proteomic approaches towards protein identification rely upon the generation of peptides through the use of specific proteases. However, for a variety of reasons, the digestion of proteins bound to planar arrays by specific proteases, such as trypsin, has proven to be difficult, at times providing little or no protein digestion at all. Additionally, should more than one protein be present on the array surface, the digestion product consists of peptides from different proteins, adding another dimension of complexity to database mining approaches. These factors have driven our group to explore alternative means of on-chip protein digestion. In this article, we describe an approach to generate peptide maps by limited acid hydrolysis. Depending upon the adsorbed protein, this method requires between 500 femtomole to 5 picomole of protein for on-chip hydrolysis. Besides generating several internal peptide fragments, limited acid hydrolysis also has the advantage of generating peptide ladders from the N- or C-terminus of the protein. From these ladders, partial primary sequence of the protein can be directly derived when analyzed by a simple laser desorption/ionization mass spectrometer. Furthermore, tandem mass spectrometry can be performed on several internal peptide fragments, thus facilitating the identification of several proteins within a mixture. Based upon the preliminary results of this work, we continue to explore the possibility of using limited acid hydrolysis to identify unknown proteins captured on ProteinChip array surfaces.

Acids↗
PubMed2001

Ikaros expression as a marker for lymphoid progenitors during zebrafish development.

The Ikaros gene encodes a transcription factor that, in mice, has been shown to be essential for the correct differentiation of B and T lymphocytes and is expressed in all cells of the lymphoid lineage, including pluripotent hematopoietic stem cells. During development in zebrafish, Ikaros expression begins in lateral mesoderm, and continues in the intermediate cell mass (ICM), which is derived from lateral mesoderm and has been shown to generate primitive hematopoietic precursors. Cells expressing Ikaros were then seen on the ventral side of the dorsal aorta, known to be a location of definitive hematopoietic precursors in birds and mammals. Ikaros-expressing cells were also found in the pharyngeal region, near the forming thymus. Later, such cells were seen in the pronephros, the site of hematopoiesis in adult fish. The timing of appearance of Ikaros-expressing cells suggests that, similar to other vertebrates, lymphocytes in the thymus arise from hematopoietic tissue located near the dorsal aorta or in the ICM.

Animals↗
PubMed2001

Concurrent expression of recombination activating genes 1 and 2 in zebrafish olfactory sensory neurons.

Each olfactory sensory neuron (OSN) expresses a single odorant receptor (OR) from a large repertoire of clustered OR genes. It has been hypothesized that OR gene regulation may involve stochastic DNA rearrangement, which in lymphocytes requires the recombination activating genes, rag1 and rag2. We have recently demonstrated that rag1 is expressed in zebrafish OSNs. Here we report that rag2, the obligate partner for rag1 function, is also expressed in OSNs and that its expression pattern mimics that of rag1. The onset of rag1 and rag2 expression preceded that of known zebrafish ORs and the number of rag1-positive OSNs corresponded with the number expressing the olfactory cyclic nucleotide-gated cation channel, an OSN marker. Zebrafish OSNs are the first example of concurrent rag expression in a nonlymphoid tissue. The expression of rag1 and rag2 in OSNs adds to the list of similarities between the olfactory and immune systems that includes monoallelic and mutually exclusive gene expression.

Animals↗
PubMed2001

Two-locus modeling of asthma in a Hutterite pedigree via Markov chain Monte Carlo.

Bayesian Markov chain Monte Carlo (MCMC) segregation analysis for asthma was performed on the whole 1,544-member Hutterite pedigree. Heterogeneous and epistatic two-locus models and complex one-locus models were investigated, with trait loci postulated to be linked to markers in regions previously found to be possibly linked to asthma or atopy. The epistatic two-locus dominant-dominant model provided the best estimates, among the models investigated, in terms of prediction of population prevalence and relative risk for sibs of the affected.

Asthma↗
PubMed2001

Development of PMMA-precoating metal prostheses via injection molding: residual stresses.

Injection molding polymethylmethacrylate (PMMA) was developed as a potential metal prosthesis precoating method for surgical implantation. However, residual stresses generated in the injection-molded PMMA affect the mechanical performance of the coating polymer. This study used plane polariscope testing to demonstrate the residual stress distribution in the injection-molded polymer. Slitting and strain gages were combined with finite element analysis to give a quantitative measure of the residual stresses. The residual stress testing indicated that the highest circumferential residual stress in the injection-molded PMMA was approximately 5 MPa in tension. Two methods, water soaking and slitting, were developed for reducing residual stresses. A series of four surgical implantation simulation processes were designed. The two methods for reducing residual stress were used in the simulations.

Animals↗
PubMed2001

Sequencing cyclic peptide inhibitors of mammalian ribonucleotide reductase by electrospray ionization mass spectrometry.

Mammalian ribonucleotide reductase (mRR) is a potential target for cancer intervention. A series of lactam-bridged cyclic peptide inhibitors (1-9) of mRR have been synthesized and tested in previous work. These inhibitors consist of cyclic and linear regions, causing their mass spectral characterization to be a challenge. We determined the fragmentation mechanism of cyclic peptides 1-9 using an ion-trap mass spectrometer equipped with an ESI source. Low-energy collision-induced dissociation of sodiated cyclic peptides containing linear branches follows a general pathway. Fragmentation of the linear peptide region produced mainly a and b ions. The ring peptide region was more stable and ring opening required higher collision energy, mainly occurring at the amide bond adjacent to the lactam bridge. The sodium ion, which bound to the carbonyl oxygen of the lactam bridge, acted as a fixed charge site and directed a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues were cleaved sequentially from the C-terminus to the N-terminus. Our findings have established a new way to sequence cyclic peptides containing a lactam bridge based on charge-remote fragmentation. This methodology will permit unambiguous identification of high-affinity ligands within cyclic peptide libraries.

Enzyme Inhibitors↗
PubMed2001

(R)-methanandamide and Delta 9-THC as discriminative stimuli in rats: tests with the cannabinoid antagonist SR-141716 and the endogenous ligand anandamide.

RATIONALE AND OBJECTIVES: (R)-methanandamide (AM-356), a metabolically more stable chiral analog of the endocannabinoid ligand anandamide, was used as a representative of fatty acid ethanolamide CB1 receptor ligands to characterize the discriminative stimulus functions of anandamides. METHODS: Rats discriminated between 10 mg/kg (R)-methanandamide and vehicle administered IP 15 min prior to session onset. Another group of rats was initially trained to discriminate between 3 mg/kg Delta9-THC and vehicle given IP 30 min prior to session onset; for anandamide testing, the animals were retrained with 1.8 and 5.6 mg/kg Delta9-THC. A two lever operant methodology (FR10) was used. RESULTS: Delta9-THC was more potent than (R)-methanandamide at both 15 and 30 min post-injection, irrespective of the training drug used. Additional tests with 10 and 18 mg/kg (R)-methanandamide suggested that the effects were declining by 1 h. The cannabinoid antagonist SR 141716 (0.3 and 1 mg/kg) produced rightward shifts in the Delta9-THC dose-response curve for Delta9-THC-appropriate responding and for (R)-methanandamide-appropriate responding (surmountable antagonism). SR-141716 (0.3 and 1 mg/kg) antagonized the ability of (R)-methanandamide to occasion either Delta9-THC-appropriate responding or (R)-methanandamide-appropriate responding. This antagonism was surmountable only at a dose of 0.3 mg/kg SR-1421716 in the (R)-methanandamide-trained rats. SR-141716 did not antagonize the rate-decreasing effects of (R)-methanandamide in either the Delta9-THC or the (R)-methanandamide trained rats. Response suppression precluded testing doses higher than 30 mg/kg (R)-methanandamide. Tests with SR-141716 (1 and 10 mg/kg) alone resulted in <3% Delta9-THC-appropriate responding. With 10 mg/kg SR-141716, response rate was significantly lower as compared to the rate observed during a vehicle test. Tests with anandamide (10 and 18 mg/kg) resulted in 41% and 85% (R)-methanandamide-appropriate responding at a 3-min pre-treatment time, but in a maximum of 15% (R)-methanandamide-appropriate responding at a longer (15 min) pre-treatment time. In the Delta9-THC (1.8 and 5.6 mg/kg) trained rats, anandamide never produced more than about 20% Delta9-THC-appropriate responding. CONCLUSION: The results add to a growing body of evidence indicating that there are both similarities and dissimilarities between classical cannabinoids such as THC and endogenous fatty acid ethanolamides.

Adjuvants, Immunologic↗
PubMed2001

Spectral inhomogeneity of photosystem I and its influence on excitation equilibration and trapping in the cyanobacterium Synechocystis sp. PCC6803 at 77 K.

Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685--687 nm within few transfer steps (300--400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4-6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30--50-ps process of photochemical trapping of the excitation by P(700). These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) approximately 200--300 cm(-1)). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (>690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.

Cyanobacteria↗
PubMed2001

Early photocycle kinetic behavior of the E46A and Y42F mutants of photoactive yellow protein: femtosecond spectroscopy.

In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I(0)) and on the excited state lifetime, whereas the rate constants for the formation of the second (I(0)( not equal)) and third (I(1)) intermediates were increased by factors of approximately 30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I(0) formation is decreased by a factor of approximately 2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of approximately 100. The structural implications of these results are discussed.

Alanine↗
PubMed2001

Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes and their corresponding phosphodiester ether lipid derivatives.

Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.

Chemistry, Pharmaceutical↗
PubMed2001

Mast cell proliferation and alterations in bFGF amount and localization are involved in the response of muscle to dystrophin deficiency in hypertrophic feline dystrophy.

To test the hypothesis that basic fibroblast growth factor and mast cells play a key role in the phenotypic differences between human dystrophinopathies and hypertrophic feline muscular dystrophy, serial sections of dystrophin-deficient, carrier and normal cat muscle biopsy specimens were examined. They were stained immunohistochemically for dystrophin and different markers of differentiation such as desmin, vimentin and utrophin. Basic fibroblast growth factor was increased in the myofibers of dystrophic cats compared to normal controls and carriers. An association of basic fibroblast growth factor with fiber regeneration and necrosis was shown. The amount of mast cells was markedly increased in muscle tissue of dystrophic cats with a clear predominance of tryptase-positive cells present in large amounts in the endomysium. Mast cells, like basic fibroblast growth factor, were concentrated in areas of muscle fiber regeneration and necrosis. Our data concerning basic fibroblast growth factor and mast cells are consistent with a highly abnormal cellular environment in feline dystrophic muscle with very high levels of basic fibroblast growth factor which is likely modulated by mast cells.

Animals↗
PubMed2001

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups

Arachidonic Acids↗
PubMed2001

Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes.

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes the reduction of many disulfide-containing substrates, plays an important role in the cellular response to oxidative stress. Trivalent arsenicals, especially methyl As that contains trivalent arsenic (MAs(III)), are potent noncompetitive inhibitors of TR purified from mouse liver. Because MAs(III) is produced in the biomethylation of As, it was postulated that the extent of inhibition of TR in cultured rat hepatocytes would correlate with the intracellular concentration of methyl As. Exposure of cultured hepatocytes to inorganic As(III) (iAs(III)), MAs(III), or aurothioglucose (ATG, a competitive inhibitor of TR activity) for 30 min caused a concentration-dependent reduction in TR activity. The estimated IC(50) was >>100 microM for iAs(III), approximately 10 microM for ATG, and approximately 3 microM for MAs(III). In hepatocytes exposed to 1 microM MAs(III) for up to 24 h, the inhibition of TR activity was maximal ( approximately 40%) after exposure for 15 min. After exposure for 3 h [when most MAs(III) has been converted to dimethyl As (DMAs)], TR activity in these cells had returned to control levels. Notably, exposure of the cell to 50 microM DMAs(III) did not affect TR activity. In hepatocytes exposed to 10 microM iAs(III) for up to 24 h, the inhibition of TR activity was progressive; at 24 h, activity was reduced approximately 35%. Following exposure to iAs(III) or MAs(III), the extent of inhibition of TR activity correlated strongly with the intracellular concentration of MAs. Taken together, these results suggest that arsenicals formed in the course of cellular metabolism of As are potent inhibitors of TR activity. In particular, MAs(III), an intermediate in the metabolic pathway, is an especially potent inhibitor of TR. Hence, the capacity of cells to produce or consume the intermediates in the pathway for As methylation may be an important determinant of susceptibility to the toxic effects of As.

Animals↗