Biomedical subjects

Bin Lin

Publications and source records attributed to Bin Lin.

10 recordsLinked to original sources

PubMed2026

Generation of a New Immunodeficient Rat Model of Retinal Degeneration With LSL TdTomato Reporter and TdTomato-Pcp2 Expression.

PURPOSE: The purpose of this study was to develop a fluorescently labeled immunodeficient retinal degenerate (RD) rat model for studying photoreceptor degeneration and transplant-host connectivity using the Cre-lox system. METHODS: We developed gene constructs for CAG-LSL-TdTomato (expressing floxed TdTomato) and Pcp2-Cre (marker for ON-bipolar cells) that were injected into rat embryos. The LSL TdTomato reporter strain, created on immunodeficient RhoS334ter-3 rats (RRRC #539), was bred to homozygosity (strain SD-Foxn1rnuTg((Rho-S334X)3,CAG-TdTomato)1010Mjsuc, RRRC #1055, "RNT"). The gene construct Pcp2-Cre was injected into Long-Evans (LE) rat embryos, resulting in two Pcp2-cre founders (strain PCP2 Cre-1105 RKI, "Pcp2"), with targeted and targeted/random insertion of the transgene. F1 offspring were bred to homozygosity and immunodeficiency. To test whether TdTomato expression can be induced in "RNT" rats expressing floxed TdTomato, retinal explants of P9 "RNT" rats were exposed to AAV-PHP.eB-hSyn-myc-Cre (AAV-Syn-Cre) virus. Homozygous rats of both strains ("RNT" and Pcp2-Cre) were crossbred to generate RD TdTomato-Pcp2 ("RTP") rats. Retinas were stained for various retinal markers. GFP-expressing rat retinas were transplanted to 6-week-old "RTP" rats and analyzed after 37 and 77 days. RESULTS: AAV-Syn-Cre induced TdTomato expression in "RNT" retinas. TdTomato-Pcp2 RD rats developed RD similar to the original Rho S334ter-3 rats. Retinas with targeted Pcp2-Cre insertion showed TdTomato in retinal interneurons, overlapping with Pcp2-staining ON bipolar cells, and cones. Retinas with random Pcp2-Cre insertion exhibited additional TdTomato in many other cells. Pcp2-TdTomato expression defined transplant-host boundaries. CONCLUSIONS: We created a unique RD rat model for studying retinal transplant connectivity which can also be used to generate RD rats with other cell-specific labels. TRANSLATIONAL RELEVANCE: This newly created rat is useful for cell therapy and retinal degeneration studies.

Animals↗

PubMed2003

Isolation and characterization of a new advanced glycation endproduct of dehydroascorbic acid and lysine.

Proteins are subject of posttranslational modification by sugars and their degradation products in vivo. The process is often referred as glycation. L-Dehydroascorbic acid (DHA), an oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. A new product of modification of lysine epsilon -amino group by DHA was discovered as a result of the interaction between Boc-Lys and dehydroascorbic acid. The chromatographic and spectral analyses revealed that the structure of the product was 1-(5-ammonio-5-carboxypentyl)-3-oxido-4-(hydroxymethyl)pyridinium. The same compound was isolated from DHA modified calf lens protein after hydrolysis and chromatographic separation. The study confirmed that L-erythrulose is an important intermediate of modification of proteins by DHA. The structure of the reported product and in vitro experiments suggested that L-erythrulose could further transform to L-threose, L-erythrose and glycolaldehyde under conditions similar to physiological. The present study revealed that the modification of epsilon -amino groups of lysine residues by DHA is a complex process and could involve a number of reactive carbonyl species.

Acetaldehyde↗

PubMed2003

Separation of the yellow chromophores in individual brunescent cataracts.

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I-V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III-V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A(330) and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development.

Aged↗

PubMed2003

Integrins regulate NMDA receptor-mediated synaptic currents.

Synapses contain high concentrations of integrins, adhesion receptors known to influence the operation of neighboring transmembrane proteins. Evidence that integrins are important for consolidation of long-term potentiation suggests that these adhesion proteins may modulate activities of synaptic glutamate receptors. The present study provides a first test of the possibility that integrins modulate synaptic N-methyl-d-aspartate (NMDA)-type glutamate receptor activities. Excitatory postsynaptic currents (EPSCs) were recorded with whole cell clamp from hippocampal slices in which AMPA-type glutamate receptors and GABA(A) receptors were pharmacologically blocked. Microperfusion of the peptide integrin ligand gly-arg-gly-asp-ser-pro (GRGDSP) caused an approximately twofold increase in the amplitude and duration of NMDA receptor-gated synaptic currents. Control peptides had no effect. Paired-pulse facilitation was unchanged, indicating that the ligand did not modify neurotransmitter release probabilities. Infusion of the Src kinase antagonist PP2 but not the control drug 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine eliminated the enhancing effect of GRGDSP. Integrins regulate Src kinases that are known to phosphorylate NMDA receptors. It is concluded that integrins act through this route to exert potent modulatory effects on the operation of NMDA receptors.

Animals↗

PubMed2002

Interactions between recording technique and AMPA receptor modulators.

Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 degrees C and with different solutions in the clamp pipette. The larger effect of cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.

Animals↗

PubMed2002

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue.

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60. Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.

Adult↗

PubMed2002

Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java, Indonesia.

In Java, Indonesia, many nutrient-poor soils are intensively reforested with Pinus merkusii (pine). Information on nutrient cycles and microorganisms involved in these cycles will benefit the management of these important forests. Here, seasonal effects on the stratification of bacterial community structure in the soil profile of a tropical pine forest are described, and differences in bacterial communities are related to chemical and physical soil parameters. Culture-independent community profiles of litter, fragmented litter and mineral soil layers were made by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA-specific polymerase chain reaction (PCR) fragments. The community profiles of the different soil layers clustered separately, correlating with significant differences in organic matter content between the three layers. The bacterial communities appeared to be stable during the wet season of 1998. The drought in 1997, caused by the El Niño climatic effect, did not influence the bacterial communities in fragmentation and mineral soil, although moisture content and other soil parameters were markedly lower than in the wet season. However, communities in litter were influenced by drought. In the litter layer, the moisture content was significantly lower than in the fragmentation and mineral layers during the dry season. A clone library was made from a litter sample taken during the wet season. Partial sequencing of 74 clones and linking the DGGE banding positions of these clones to bands in the DGGE profile of the sample from which the clone library was derived showed considerable bacterial diversity. Alpha-proteobacteria (40.5% of the clones, of which 57% belonged to the Rhizobium-Agrobacterium group) and high-G+C content, Gram-positive bacteria (36.5%) dominated the clone library.

Bacteria↗

PubMed2002

Long-term potentiation alters the modulator pharmacology of AMPA-type glutamate receptors.

Changes in the biophysical properties of AMPA-type glutamate receptors have been proposed to mediate the expression of long-term potentiation (LTP). The present study tested if, as predicted from this hypothesis, AMPA receptor modulators differentially affect potentiated versus control synaptic currents. Whole cell recordings were collected from CA1 pyramidal neurons in hippocampal slices from adult rats. Within-neuron comparisons were made of the excitatory postsynaptic currents (EPSCs) elicited by two separate groups of Schaffer-collateral/commissural synapses. LTP was induced by theta burst stimulation in one set of inputs; cyclothiazide (CTZ), a drug that acts on the desensitization kinetics of AMPA receptors, was infused 30 min later. The decay time constants of the potentiated EPSCs prior to drug infusion were slightly, but significantly, shorter than those of control EPSCs. CTZ slowed the decay of the EPSCs, as reported in prior studies, and did so to a significantly greater degree in the potentiated synapses. Additionally, infusion of CTZ resulted in significantly greater effects on amplitude in potentiated pathways as compared with control pathways. The interaction between LTP and CTZ was also obtained in a separate set of experiments in which GABA receptor antagonists were used to block inhibitory postsynaptic currents. Additionally, there was no significant change in paired-pulse facilitation in the presence of CTZ, indicating that presynaptic effects of the drug were negligible. These findings provide new evidence that LTP modifies AMPA receptor kinetics. Candidates for the changes responsible for the observed effects of LTP were evaluated using a model of AMPA receptor kinetics; a simple increase in the channel opening rate provided the most satisfactory match with the LTP data.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2002

Microbial aspects of anaerobic BTEX degradation.

Combined with conventional methods, developments in both geochemical (delineation of redox processes) and molecular microbial methods (analysis of 16S rDNA genes and functional genes) have allowed us to study in details microorganisms and genes involved in the anaerobic degradation of benzene, toluene, ethylbenzene and xylene (BTEX) under specific redox conditions. This review summarizes recent research in this field. The potential for anaerobic BTEX degradation is widely spread. Specific groups of microorganisms appear to be involved in degradation under different redox conditions. Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulfate reducing conditions. The information so far obtained on biochemistry and molecular genetics of BTEX degradation indicates that each BTEX compound is funneled into the central benzyol-CoA pathway by a different peripheral pathway. The peripheral pathways of per BTEX compound show similarities among different physiological groups of microorganisms. We also describe how knowledge obtained on the microbial aspects of BTEX degradation can be used to enhance and monitor anaerobic BTEX degradation.

Bacteria, Anaerobic↗

PubMedDate not supplied

Expression and distribution of ionotropic glutamate receptor subunits on parasol ganglion cells in the primate retina.

The response properties of postreceptoral sensory neurones are determined by the properties of their input neurones, by intrinsic membrane properties, and by the properties of neurotransmitter receptors on the soma and dendritic tree. We previously showed that inhibitory neurotransmitter (GABA(A) and glycine) receptors on a well-characterised sensory neurone, the parasol ganglion cell in the primate retina, are segregated towards the distal part of the dendritic tree. Here we studied the distribution of excitatory ionotropic glutamate receptor subunits on the dendrites of parasol cells in the retina of a New World monkey, the marmoset, Callithrix jacchus. Individual ganglion cells were intracellularly injected in an in vitro retinal wholemount preparation. Ionotropic glutamate receptor subunits. including AMPA (GluR1-4), kainate (GluR6/7), NMDA (NR1C2') subunits, and the orphan receptors delta1 and delta2 were visualized with immunocytochemical methods. Immunoreactive puncta that colocalized with the dendrites of ganglion cells were analyzed using standard and/or confocal light microscopy. Colocalized puncta were present on parasol dendrites for all subunits studied, but their density was much lower (approximately 1/5) than previously reported for inhibitory (GABA and glycine) receptors. Segregation of the glutamate receptor clusters (GluR1, GluR6/7 subunits) to the peripheral dendrites was less marked than that shown for GABA and glycine receptor clusters. No sign of segregation of colocalized puncta to the peripheral part of the dendritic field was seen with antibodies to the GluR2, GluR2/3, GluR4, delta1/2, or NR1C2' subunits. The results suggest that although there is diverse expression of glutamate receptor subtypes, the glutamatergic synapses form only a small proportion of the total synaptic input to primate ganglion cells. They further suggest that the processes which control distribution of excitatory and inhibitory synapses on the dendritic field of ganglion cells are, at least to some extent, independent.

Animals↗