Investigation of relationships between collagens, elastin and proteoglycans in bovine thoracic aorta by immunofluorescence techniques

Types I, III and V collagens and proteoglycan were localized in the aorta by indirect immunofluorescence techniques. Type I collagen was more prominent in media and adventitia than in intima while type III collagen predominated in intima and media but appeared less significant in adventitia. Type V collagen was observed in intima and media only and was seen surrounding smooth muscle cells. Type I collagen was located between elastic fibres but type III collagen appeared to envelop the fibres, suggesting an interaction between elastic fibres and type III collagen. Pretreatment of sections with testicular hyaluronidase caused no changes in staining for type I collagen, but adventitial areas showed increased staining for type III collagen. After digestion with chondroitinase ABC, intimal and medial areas showed increased staining for type III collagen. Therefore, type III collagen forms stronger interactions with proteoglycans and hyaluronic acid than does type I collagen and type III collagen in adventitia is largely masked by hyaluronic acid, while type III collagen in intima and media is associated with proteoglycan. Thus, type III collagen is a more significant component of adventitia than previously recognized. Proteoglycan was also partly localized along elastic fibres. It is, therefore, suggested that elastic fibres are coated with type III collagen, which itself is coated with proteoglycan.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

New sites of methylcytosine-rich DNA detected on metaphase chromosomes

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.     

Type V collagen in splenic reticular fibers of the macaque monkey

The distribution of type I, III and V collagens in the monkey spleen was examined by indirect immunofluorescent microscopy and immunoelectron microscopy, and compared with that of reticular fibers revealed by a silver impregnation method. Type I collagen was localized on reticular fibers in the white pulps and on coarse reticular fibers in the splenic cords. Type III collagen was localized on the reticular fibers in the white pulps, and on the coarse reticular fibers and a limited number of fine reticular fibers, in the splenic cords. The anti-type V collagen antibody reacted with annular reticular fibers around the splenic sinuses, as well as with the reticular fibers in the white pulps and with the coarse and fine reticular fibers in the splenic cords. Thus, the distribution pattern of fibers that reacted with the anti-type V collagen antibody was very similar to that of the reticular fibers revealed by the silver impregnation method. Electron-microscopically, the fine reticular fibers in the splenic cords were composed of collagen fibrils, 30-50 nm in diameter, and amorphous substances. They were covered by reticular cell processes. By immunoperoxidase labeling with the anti-type V collagen antibody, electron-dense reaction products were found over the collagen fibrils with a banding pattern. These results indicate that type V collagen is an indispensable component of the reticular fibers.     

Fluorescence characterization of type I collagen from normal and silicotic rats and its quenching dynamics induced by hypocrellin B

In this paper, we studied the quenching mechanism of intrinsic fluorescence of type I collagen by a new type photosensitizer and fluorescence quencher, hypocrellin B (HB). It was indicated that type I collagen can emit Tyr-intrinsic fluorescence with the excitation wavelength of Tyr (λ_ex = 269 nm). Its fluorescence decay conform to the triexponential rule of the fluorescence lifetime. The intrinsic fluorescence of type I collagen can be effectively quenched by HB through a process of charge and energy transference, which is involved in the collisional quenching, the dipolar inducement, and the formation of exciplex between HB and excited fluorophores of collagen. The fluorescence quenching would be weakened by higher ionic environments. The fluorescence emission and its quenching rate of abnormal silicotic collagen show falling trends, implying its much weakened potential of charge and energy transference, and its lessen bioelectric activities. In conclusion, the bioelectric properties of collagen depends on the perfect order of its molecular structure and orderly intramolecular and intermolecular interactions, which is important in its performing normal physiological functions. It is also demonstrated that the fluorescence quenching technique, using HB as a quencher, is truly an effectively method for biomolecular studies. © 1997 John Wiley & Sons, Inc. Biopoly 42: 219–226, 1997     

Connective tissue of rat lung. II: Ultrastructural localization of collagen types III, IV, and VI

We localized collagen types III, IV, and VI in normal rat lung by light and electron immunohistochemistry. Type IV collagen was present in every basement membrane examined and was absent from all other structures. Although types III and VI had a similar distribution, being present in the interstitium of major airways, blood vessels, and alveolar septa, as in other organs, they had different morphologies. Type III collagen formed beaded fibers, 15-20 nm in diameter, whereas type VI collagen formed fine filaments, 5-10 nm in diameter. Both collagen types were found exclusively in the interstitium, often associated with thick (30-35 nm) cross-banded type I collagen fibers. Occasionally, type III fibers and type VI filaments could be found bridging from the interstitium to the adventitial aspect of some basement membranes. Furthermore, the association of collagen type VI with types I and III and basement membranes suggests that type VI may contribute to integration of the various components of the pulmonary extracellular matrix into a functional unit.     

Impairment of myocardial calcium homeostasis by antibodies against the adenine nucleotide translocator.

The adenine nucleotide translocator (ANT) is an autoantigen in myocarditis and dilated cardiomyopathy. Carrier-specific antibodies impair myocardial energy metabolism and heart function. They cross-react with a myolemmal calcium channel and alter calcium fluxes in isolated myocytes. To test whether antibodies against the ANT can alter calcium homeostasis in intact hearts, guinea pigs were immunized with the carrier protein and their isolated hearts loaded with the intracellular calcium indicator INDO-1. The diastolic and systolic ratios of fluorescence signals at 410 nm and 510 nm (emission wavelengths of the calcium-bound and calcium-free indicator), 'd-s410/510', were measured by excitation at 364 nm. This index of the transient calcium concentration associated with the contraction cycle correlated with the external heart work (EHW) in non-immunized controls. EHW of immunized animals was lower (76 +/- 62 vs 153 +/- 47 mJ/g/min in controls, p < 0.005) and the amplitude of d-s410/510 was elevated (27.6 +/- 4.1% of the average ratio of the whole heart cycle vs 21.7 +/- 1.2% in controls, p < 0.005) and essentially independent of EHW. Isoproterenol stimulation increased EHW in all hearts but d-s410/510 was hightened in control hearts, only. Thus, a disorder between cytosolic calcium transients and work was recorded in hearts from guinea pigs immunized with the ANT. It may contribute to an immunopathic mechanism of heart failure subsequent to myocarditis.     

DNA-templated formation and luminescence of diphenylacetylene dimeric and trimeric complexes

We report on spectral features for two and three diphenylacetylene chromophores aligned in close proximity in aqueous solution by self assembly of attached oligonucleotide arms. Two duplex systems were examined in detail. One was formed by hybridization (Watson-Crick base pairing) of two oligonucleotide 10-mers, each containing the diphenylacetylene insert. The other was generated by self-folding of a 36-mer oligonucleotide containing two diphenylacetylene inserts. The triplex system was obtained by hybridization (Hoogsteen base pairing) of a 16-mer oligonucleotide diphenylacetylene conjugate to the folded 36-mer hairpin. Formation of duplex and triplex entities from these conjugates was demonstrated experimentally by thermal dissociation and spectroscopic studies. The UV and CD spectra for the duplex systems exhibit bands in the 300-350 nm region attributable to exciton coupling between the two chromophores, and the emission spectra show a strong band centered at 410 nm assigned to excimer fluorescence. Addition of the third strand to the hairpin duplex has little effect on the CD spectrum in the 300-350 nm region, but leads to a negative band at short wavelengths characteristic of a triplex and to a strongly enhanced band at 410 nm in the fluorescence spectrum. The third strand alone shows a broad fluorescence band at approximately 345-365 nm, but this band is virtually absent in the triplex system. A model for the triplex system is proposed in which two of the three aligned diphenylacetylenes function as a ground state dimer that on excitation gives rise to the exciton coupling observed in the UV and CD spectra and to the excimer emission observed in the fluorescence spectrum. Excitation of the third chromophore results in enhanced excimer fluorescence, as a consequence of energy transfer from the locally excited singlet of one chromophore to the ground state dimer formed by the other two chromophores.     

Spectroscopic studies on origination of the peak at 730 nm in delayed fluorescence of chloroplasts.

The origination of the peak at 730 nm in the delayed fluorescence (DF) spectrum of chloroplasts was studied using various optical analysis methods. The DF spectrum showed that the main emission peak was at about 685 nm, with a small shoulder at 730 nm when the chloroplast concentration was < 7.8 microg/mL. The intensity of the peak at 685 nm decreased, while the intensity of the peak at 730 nm increased, when the chloroplast concentrations were increased from 7.8 to 31.2 microg/mL. With the concentration increasing, the peak at 730 nm became dominant while the peak at 685 nm finally disappeared. The DF decay kinetic curves showed that the intensity of the peak at 730 nm decayed as the same speed as the intensity of the peak at 685 nm during the entire relaxation process (0.5-30.5 s). With the excitation wavelength at 685 nm, the emission intensity was stronger in the excitation spectrum at 730 nm. The absorption spectrum demonstrated that the ratio A(685):A(730) remained almost constant when the chloroplast concentration increased. The results suggest that the peak at 730 nm appearing in DF is mainly contributed by the fluorescence of photosystem I (PSI), generated by the re-absorption of 685 nm band DF.     

Innervation of the intestine in the bivalve mollusc Chione stutchburyi

Summary The innervation of the gut of the venerid bivalve mollusc, Chione stutchburyi, has been examined by fluorescence histochemistry, electron microscopy and autoradiography. Specific green and yellow varicose fluorescent fibres indicate the presence of dopaminergic and serotonergic axons, respectively. Three different types of axons can be distinguished by the morphological characteristics of their vesicles. Type I axons contain predominantly small granular vesicles (average diameter 65 nm), Type II axons possess large granular vesicles (average diameter 100 nm) and Type III axons contain large opaque vesicles (average diameter 150 nm). The granular vesicles in both Types I and II axons react positively to dichromate, and their granularity is reduced by reserpine indicating that they are monoaminergic. Only Type I axons accumulate tritiated dopamine and are selectively damaged by 6-hydroxydopamine. It is concluded that Type I axons are dopaminergic. Type II axons are serotonergic: they alone take up tritiated 5-hydroxytryptamine, and 5,7-dihydroxytryptamine selectively causes degenerative changes in these axons. Type III axons contain an unidentified neurotransmitter substance. The large opaque vesicles of these axons do not react to dichromate and are unaffected by reserpine, 6-hydroxydopamine or 5,7-dihydroxytryptamine.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490?nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max) ≈?490 to λ(max) ≈?476?nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472?nm, whereas other Vibrio strains emit light with a peak at around 482?nm. Therefore, we investigated the mechanism underlying this blue shift in V.?azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V.?azureus.     

Polarized site-selective fluorescence spectroscopy of the long-wavelength emitting chlorophylls in isolated Photosystem I particles of Synechococcus elongatus

Isolated trimeric Photosystem I complexes of the cyanobacterium Synechococcus elongatus have been studied with absorption spectroscopy and site-selective polarized fluorescence spectroscopy at cryogenic temperatures. The 4 K absorption spectrum exhibits a clear and distinct peak at 710 nm and shoulders near 720, 698 and 692 nm apart from the strong absorption profile located at 680 nm. Deconvoluting the 4 K absorption spectrum with Gaussian components revealed that Synechococcus elongatus contains two types of long-wavelength pigments peaking at 708 nm and 719 nm, which we denoted C-708 and C-719, respectively. An estimate of the oscillator strengths revealed that Synechococcus elongatus contains about 4–5 C-708 pigments and 5–6 C-719 pigments. At 4 K and for excitation wavelengths shorter than 712 nm, the emission maximum appeared at 731 nm. For excitation wavelengths longer than 712 nm, the emission maximum shifted to the red, and for excitation in the far red edge of the absorption spectrum the emission maximum was observed 10–11 nm to the red with respect to the excitation wavelength, which indicates that the Stokes shift of C-719 is 10–11 nm. The fluorescence anisotropy, as calculated in the emission maximum, reached a maximal anisotropy of r=0.35 for excitation in the far red edge of the absorption spectrum (at and above 730 nm), and showed a complicated behavior for excitation at shorter wavelengths. The results suggest efficient energy transfer routes between C-708 and C-719 pigments and also among the C-719 pigments.Abbreviations Chl chlorophyll - FWHM full width at half maximum - PS I Photosystem I     

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, alpha-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.     

A new silver nanochain SERS analytical platform to detect trace hexametaphosphate with a rhodamine S molecular probe

Using AgNO₃ as the precursor, stable silver nanochain (AgNC) sols, orange‐red in color, were prepared using hydrazine hydrate. A strong surface plasmon resonance Rayleigh scattering (RRS) peak occurred at 420 nm plus two surface plasmon resonance (SPR) absorption peaks at 410 nm and 510 nm. Rhodamine S (RhS) cationic dye was absorbed on the as‐prepared AgNC substrate to obtain a RhS–AgNC surface‐enhanced Raman scattering (SERS) nanoprobe that exhibited a strong SERS peak at 1506 cm^–1 and a strong RRS peak at 375 nm. Upon addition of the analyte sodium hexametaphosphate (HP), it reacted with RhS, which resulted in a decrease in the SERS and RRS peaks that was studied in detail. The decreased SERS and RRS intensities correlated linearly with HP concentration in the range of 0.0125–0.3 µmol/L and 0.05–1.0 µmol/L, with a detection limit of 6 nmol/L and 20 nmol/L HP respectively. Due to advantages of high sensitivity, good selectivity and simple operation, the RhS molecular probes were used to determine HP concentration in real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum

We examined the ultrastructural localization of collagens Type I, V, VI and of procollagen Type III in decalcified and prefixed specimens of the periodontal ligament and cementum, by immunoelectron microscopy using ultra-thin cryostat sections. Immunostaining for collagen Type I was pronounced on the major cross-striated fibrils entering cementum and in cementum proper, whereas staining for procollagen Type III was almost exclusively observed on the major fibrils in the periodontal ligament situated more remote from cementum. Reactivity for collagen Type V was limited to aggregated, unbanded filamentous material of about 12 nm diameter that was found mainly in larger spaces between bundles of cross-striated collagen fibrils and occasionally on single microfibrils that apparently originated from the ends of the major collagen fibrils, which may support the concept of this collagen as a component of core fibrils. Collagen Type VI was present as microfilaments appearing to interconnect single cross-striated fibrils. In the densely packed fibril bundles of the periodontal ligament, no collagen type VI was detected. Neither Type V or Type VI collagen was observed in cementum.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

CdSe and CdSe/CdS core–shell QDs: New approach for synthesis,investigating optical properties and application in pollutant degradation

In this work, CdSe quantum dots (QDs) were synthesized by a simple and rapid microwave activated approach using CdSO₄, Na₂SeO₃ as precursors and thioglycolic acid (TGA) as capping agent molecule. A novel photochemical approach was introduced for the growth of CdS QDs and this approach was used to grow a CdS shell around CdSe cores for the formation of a CdSe/CdS core–shell structure. The core–shells were structurally verified using X‐ray diffraction, transmission electron microscopy and FTIR (Fourier‐transform infrared (FTIR)) spectroscopy. The optical properties of the samples were examined by means of UV–Vis and photoluminescence (PL) spectroscopy. It was found that CdS QDs emit a broad band white luminescence between 400 to 700 nm with a peak located at about 510 nm. CdSe QDs emission contained a broad band resulting from trap states between 450 to 800 nm with a peak located at 600 nm. After CdS shell growth, trap states emission was considerably quenched and a near band edge emission was appeared about 480 nm. Optical studies revealed that the core–shell QDs possess strong ultraviolet (UV) ? visible light photocatalytic activity. CdSe/CdS core–shell QDs, showed an enhancement in photodegradation of Methyl orange (MO) compared with CdSe QDs.