578.2 nm激光照射对兔视网膜的损伤效应研究

研究578.2 nm激光照射对兔视网膜的作用特点,以新西兰白兔5只10眼为实验对象,铜蒸汽激光(578.2 nm)通过裂隙灯照射兔视网膜后极部,照射时间为100 s,光斑直径为2 mm,照射剂量分别为60 J/cm2、80 J/cm2、100 J/cm2、120 J/cm2、160 J/cm2、200 J/cm2,每组4个光斑。照后1 h及24 h进行眼底照相及光镜观察。照光后可见,随激光功率密度的增加,兔视网膜的损伤也逐渐加重,并且照后24 h的损伤要重于照后1h。80 J/cm2和60 J/cm2在照后1 h和24 h均未发现明显改变。578.2 nm激光照射白兔后的主要病理学改变位于脉络膜。因此,以578.2 nm激光作为光动力治疗眼底疾病的光源时,照射剂量不宜超过80 J/cm2。     

Density of rabbits (

The density of rabbits (Oryctolagus cuniculus) in the modified tussock grasslands of the Mackenzie Basin, South Island, New Zealand, in August-September 1991 was determined within 26 I-ha quadrats spread over 1000 ha. The area was poisoned with 1080- carrot baits and dead and live rabbits counted. The overall kill rate was 93%. Wide variability in rabbit densities amongst the quadrats was correlated with burrow density, but vegetation was not a significant predictor of rabbit numbers. High density quadrats were not all spatially clumped together. Variation amongst quadrats of 0-43% of rabbits dying underground shows that searching burrows as well as the surface will provide the most accurate rabbit densities. Poisoning efficacy was greater than or equal to 90% in 77% of the quadrats, but two low- density quadrats recorded kill rates <70%. Nine per cent of carcasses had been partly eaten by predators, suggesting a potential for the predators to be killed during rabbit control operations via secondary poisoning. The average density of 19 rabbits per ha confirms the classification of this area as a moderate to high rabbit-prone zone, with the rabbit population imposing grazing pressure equivalent to at least 1-2 sheep per ha at the beginning of spring. Sustainable agriculture cannot be attained in these semi-arid regions of New Zealand without cheap, widespread and effective rabbit control.     

Light and electron microscopic studies on the localization of steroid-binding protein (SBP) in rabbit spermatozoa

Light (fluorescence) and electron microscopic studies were carried out to localize steroid-binding protein (SBP) in rabbit spermatozoa. Both nonpermeabilized and permeabilized (with Tween 20, saponin, or cold acetone) spermatozoa showed fluorescence following treatment with antirabbit SBP (anti-rSBP) and subsequently with rabbit antisheep immunoglobulin G-fluorescein isothiocyanate. While the ejaculated spermatozoa were positive, epididymal sperm were observed to be negative. Although the pattern of localization of rSBP was variable, the occurrence of a negative equatorial region as well as the presence of an intense positive spherical profile ("spot") at the junction of the head and midpiece were notably consistent. The intensity of labeling with the probe, both at light and electron microscopic level, was maximal following permeabilization with cold acetone. A possible role of SBP as a steroid carrier protein across the plasma membrane of the sperm has been suggested.     

Optimizing dye-ligand density with molecular analysis for affinity chromatography of rabbit muscle L-lactate dehydrogenase

Ligand density is an important factor in determining the binding capacity and separation efficiency for affinity chromatography. A molecular analysis method based on the three-dimensional structure of protein and protein-ligand interactions was introduced to optimize the dye-ligand density for target protein separation. Expanded-bed adsorption (EBA) of L-lactate dehydrogenase (LDH) from rabbit muscle crude extract with Procion Red HE-3B as the dye-ligand was used as the model. After the analysis of LDH three-dimensional molecular structure and dye-protein interaction modes, the rational dye-ligand distance was predicted at about 20 A for efficiently binding LDH. A series of dye-ligand adsorbents with different ligand densities were prepared, and the isotherm adsorption equilibria of LDH were measured. High adsorption capacity of LDH was achieved at about 1600 U/mL adsorbent. Packed-bed chromatography was performed, and the elution effects were investigated. Finally, an EBA process was achieved to capture the LDH directly from rabbit muscle crude extract. The method established in the present work could be expanded to guide the screening of ligand density for other affinity chromatographic processes.     

Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus

A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum from a rabbit immunized with the recombinant protein recognized the 27.5 kDa viral envelope protein of WSSV isolated from different geographic regions. The antiserum did not recognize any of the other known WSSV structural proteins. A sensitive immunodot assay for WSSV was developed using the specific rabbit polyclonal antiserum.     

Topographical mapping of sites of enhanced HRP permeability in the normal rabbit aorta.

The spatial distribution of sites of enhanced permeability to the macromolecule horseradish peroxidase (HRP) in the normal rabbit aorta after one min circulation was studied using image analysis. These sites, referred to as "HRP spots," exhibit a nonuniform distribution that is qualitatively similar in all rabbits studied. The density of HRP spots is highest in the aortic arch, decreases distally, reaches a minimum in the lower descending thoracic aorta, and then increases again in the abdominal aorta. The region of highest spot density follows a clockwise helical pattern in the aortic arch and outside the arch occurs in streaks largely oriented in the bulk flow direction. The streaks in the abdominal aorta localize along the anatomical right lateral wall and occasionally along the left lateral wall proximal to the celiac artery and along the ventral wall between the celiac and superior mesenteric arteries. The density of spots is high in the immediate vicinity of aortic ostia with the most elevated density being distal to ostia in most cases. At a short distance from the ostium edge of the celiac and superior mesenteric branches the proximal density is comparably high, and no preferred spot orientation is observed around the brachiocephalic vessel. These results are consistent with an influence of localizing factors such as detailed hemodynamic phenomena and/or arterial wall structural and/or functional variations.     

Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma.

A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.     

Isolation of inclusion bodies from rabbit lung parenchyma

The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.     

Lysosomes from rabbit type II cells catabolize surfactant lipids

The role of a lysosome fraction from rabbit type II cells in surfactant dipalmitoylphosphatidylcholine (DPPC) catabolism was investigated in vivo using radiolabeled DPPC and dihexadecylphosphatidylcholine (1, 2-dihexadecyl-sn-glycero-3-phosphocholine; DEPC), a phospholipase A(1)- and A(2)-resistant analog of DPPC. Freshly isolated type II cells were gently disrupted by shearing, and lysosomes were isolated with Percoll density gradients (density range 1.0591-1.1457 g/ml). The lysosome fractions were relatively free of contaminating organelles as determined by electron microscopy and organelle marker enzymes. After intratracheal injection of rabbits with [(3)H]DPPC and [(14)C]DEPC associated with a trace amount of natural rabbit surfactant, the degradation-resistant DEPC accumulated 16-fold compared with DPPC in lysosome fractions at 15 h. Lysosomes can be isolated from freshly isolated type II cells, and lysosomes from type II cells are the primary catabolic organelle for alveolar surfactant DPPC following reuptake by type II cells in vivo.     

Predictors of clutch predation of a globally significant avifauna in New Zealand's braided river ecosystems

Predation by introduced mammals is decimating New Zealand's indigenous fauna. Understanding factors that influence this process allows resources for predator control to be applied with maximum effect. This study examines how predation of a secondary prey species (a relatively common but declining native plover, the banded dotterel Charadrius bicinctus ) varied with reductions in abundance of a major prey source (rabbits), kill-trapping of predators, nest density and habitat complexity. Banded dotterels mostly nest in open braided riverbeds alongside a number of endemic threatened species. We measured the fate of 753 dotterel clutches exposed to predation by cats, ferrets and hedgehogs. We found key times and places of high predation risk. Immediately after widespread reduction in rabbit populations by rabbit haemorrhagic disease (RHD), clutch predation rates were almost as high (mean, 50%) as those recorded during past rabbit poisoning programmes (mean, 57%). Both rates were significantly higher than the mean predation rate of 22% without rabbit control, suggesting a shift in predator diet immediately after rabbit population declines. Unlike after rabbit poisoning, clutch predation rate remained high in the years after RHD. Other patterns observed included higher clutch predation rate where nest density was lower, suggesting that predation can potentially cause local extinction. Clutch predation was also higher along riverbed margins where vegetation was dense. There was equivocal evidence for an effect of predator kill-trapping on clutch predation rate. Management strategies that could potentially reduce clutch predation risks include focusing predator mitigation measures during periods of rabbit decline, maintaining them for more than one breeding season if the rabbit declines are widespread (e.g. RHD epidemics), and applying greater effort at sites with relatively low nest density and along riverbed margins where predator use is more frequent.     

Immunofluorescence revealed the presence of NHE-1 in the nuclear membranes of rat cardiomyocytes and isolated nuclei of human, rabbit, and rat aortic and liver tissues

Using immunofluorescence and 3-dimensional confocal microscopy techniques, the present study was designed to verify if NHE-1 is present at the level of the nuclear membrane in cells that are known to express this type of exchanger. Nuclei were isolated from aortic tissues of adult human, rabbit, and rats, as well as from liver tissues of human fetus, and adult rabbit and rat. In addition, cultured ventricular cardiomyocytes were isolated from 2-week-old rat. Our results showed the presence of NHE-1 in isolated nuclei of aortic vascular smooth muscle and liver of human, rabbit, and rat. NHE-1 seems to be distributed throughout the isolated nucleus and more particularly at the level of the nuclear membranes. The relative fluorescence density of NHE-1 was significantly higher (p < 0.05) in isolated liver nuclei of human, when compared with those of rabbit and rat. However, in isolated nuclei of aortic vascular smooth muscle, the relative fluorescence density of NHE-1 was significantly (p < 0.001) higher in the rabbit when compared with human and rat. In cultured rat ventricular cardiomyocytes, NHE-1 fluorescent labeling could be easily seen throughout the cell, including the nucleus, and more particularly at both the sarcolemma and the nuclear membranes. In rat cardiomyocytes, the relative fluorescence density of NHE-1 of the sarcolemma membrane, including the cytosol, was significantly lower than that of the whole nucleus (including the nuclear envelope membranes). In conclusion, our results showed that NHE-1 is present at the nuclear membranes and in the nucleoplasm and its distribution and density may depend on cell type and species used. These results suggest that nuclear membranes' NHE-1 may play a role in the modulation of intranuclear pH.     

Differential analysis of D-beta-Asp-containing proteins found in normal and infrared irradiated rabbit lens

Although proteins are generally composed of l-alpha-amino acids, d-beta-aspartic acid (Asp)-containing proteins have been reported in various elderly tissues. Our previous study detected several d-beta-Asp-containing proteins in a rabbit lens derived from epithelial cell line by Western blot analysis of a 2D-gel using a polyclonal antibody that is highly specific for d-beta-Asp-containing proteins. The identity of each spot was subsequently determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Ms-Fit online database searching algorithm. In this study, we discovered novel d-beta-Asp-containing proteins from rabbit lens. The results indicate that beta-crystallin A3, beta-crystallin A4, beta-crystallin B1, beta-crystallin B2, beta-crystallin B3, gamma-crystallin C, gamma-crystallin D, and lambda-crystallin in rabbit lens contain d-beta-Asp residues. Furthermore, the occurrence of d-beta-Asp residues increases with infrared ray (IR) irradiation. Additionally, some d-beta-Asp-containing proteins only appear after IR irradiation. One such protein is the alpha-enolase, which shows homology to tau-crystallin.     

Purification and properties of rabbit liver phosphorylase phosphatase.

A procedure for the purification of rabbit liver phosphorylase phosphatase is described. The specific activity of the preparation is 2,100 units/mg of protein, representing a 25,000-fold purification. During the initial steps of the purification a large activation of enzyme activity was observed. The molecular weight of the purified enzyme was estimated by Sephadex G-75 chromatography to be 35,000, and by sucrose density ultracentrifugation to be 34,000 (2.9 S). On Na dodecyl-SO4 polyacrylamide disc gel electrophoresis a single component with a molecular weight of 34,000 was observed. The pH optimum is 6.9 to 7.4, and the Km for rabbit muscle phosphorylase alpha is 2 muM. The same procedure is also applicable to the extensive purification of phosphorylase phosphatase from rabbit muscle.     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Quantitative analysis of two-dimensional electrophoretograms.

A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.     

Evolution of a sexually dimorphic trait in a broadly distributed topminnow (Fundulus olivaceus)

Understanding the interaction between sexual and natural selection within variable environments is crucial to our understanding of evolutionary processes. The handicap principle predicts females will prefer males with exaggerated traits provided those traits are indicators of male quality to ensure direct or indirect female benefits. Spatial variability in ecological factors is expected to alter the balance between sexual and natural selection that defines the evolution of such traits. Male and female blackspotted topminnows (Fundulidae: Fundulus olivaceus) display prominent black dorsolateral spots that are variable in number across its broad range. We investigated variability in spot phenotypes at 117 sites across 13 river systems and asked if the trait was sexually dimorphic and positively correlated with measures of fitness (condition and gonadosomatic index [GSI]). Laboratory and mesocosm experiments assessed female mate choice and predation pressure on spot phenotypes. Environmental and community data collected at sampling locations were used to assess predictive models of spot density at the individual, site, and river system level. Greater number of spots was positively correlated with measures of fitness in males. Males with more spots were preferred by females and suffered greater mortality due to predation. Water clarity (turbidity) was the best predictor of spot density on the drainage scale, indicating that sexual and natural selection for the trait may be mediated by local light environments.     

A Comparison of the Evolutionary Distribution of the Two Neuroendocrine Markers, Neurone-Specific Enolase and Protein Gene Product 9.5

Abstract: One- and two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting has been used to examine the phylogenetic distribution of the two neuronal and neuroendocrine proteins, neurone-specific enolase and protein gene product 9. 5 , in animal brains. A new immunoblotting procedure was used in which complex two-dimensional patterns of brain proteins were transferred to nitrocellulose paper simultaneously with the Coomassie Blue stain. This produced a copy of the blue spot pattern against which brown protein spots reacting in a specific antibody-immunoperoxidase procedure could be identified unequivocally. Extracts of human, bovine, sheep, rabbit, rat, guinea-pig, chicken, trout, and frog brains were examined. Proteins cross-reacting with antisera to the human forms of both proteins could be demonstrated in all species examined. This suggests that proteins corresponding to neurone-specific enolase and protein gene product 9.5 could have evolved at least 400 million years ago and have been highly conserved throughout evolution.     

A combined oligonucleotide and protein microarray for the codetection of nucleic acids and antibodies associated with human immunodeficiency virus, hepatitis B virus, and hepatitis C virus infections

A multiplexed assay based on the codetection of nucleic acids and antibodies in human serum infected by human immunodeficiency virus (HIV), hepatitis B virus (HBV) or hepatitis C virus was proposed. The combined immuno- and oligosorbent array (CombOLISA) microarray is prepared in 96-well standard microplates by spotting (1). nucleic probes specific for a virus genome, (2). viral proteins for the capture of serum antibodies, and (3). nonspecific proteins for verifying specificity. Experimental assay conditions were optimized so that both DNA hybridization and immunological reactions can be achieved simultaneously in the same well and buffer and all at the same temperature. A generic detection system based on the precipitation of an insoluble colorimetric substrate in the presence of enzyme-labeled antibodies or streptavidin was proposed. The optical density of each spot was correlated to the corresponding analyte concentration. The influence of critical parameters on CombOLISA performance such as serum concentration was studied. Calibration curves and sensitivity thresholds were established for each parameter. Serial dilutions of serum were correlated to results obtained with validated immunoassay platforms such as a microplate enzyme-linked immunosorbent assay or the VIDAS automat. Also, several HIV- and HBV-infected serum samples were tested independently by CombOLISA and VIDAS. Coefficients of variation for genomic and proteomic parameters vs spot density were below 15%.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Electron microscopical analysis of surface charge labelling density at various stages of the erythroid line

Summary With use of the positively charged, colloidal ferric oxide labelling technique for electron microscopy of sections of rabbit marrow, a reduction in labelling density on the surface of differentiating red cell precursors was demonstrated. The number of iron particles per unit length of membrane was counted. A progressive diminution in labelling density follows cell division, reaching a minimum in the orthochromatic erythroblast, from which the nucleus is expelled. A slight increase in charge density is noted in the reticulocyte, and further increase is observed with its maturation to the erythrocyte. The results indicate that biosynthesis of n-acetyl neuraminic acid stops at the earliest recognizable stage of erythroid differentiation.The Patrick E. Gorman Professor of Biological Ultrastructure.