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Cloning and sequencing of the cDNA for the light subunit of human liver gamma-glutamylcysteine synthetase and relative mRNA levels for heavy and light subunits in human normal tissues.

We have cloned and sequenced a full length cDNA for the light subunit of human liver gamma-glutamylcysteine synthetase (GCS1). The GCS1 cDNA consists of 1628 bp, containing an open reading frame of 822 bp which encodes a protein of 274 amino acids having a calculated M(r) of 30,729 Daltons. Human liver GCS1 shares 91% and 96% sequence homology at the nucleotide and amino acid levels, respectively, with its rat counterpart (Huang, Anderson and Meister, J. Biol. Chem. 268:20578-20583, 1993). Transcripts of 1.4 and 4.1 kb in length were detected by Northern blot analysis of mRNA isolated from each of sixteen different human tissues. Relative expression of the two GCS1 RNA transcripts was highly tissue-dependent. High steady-state levels of the smaller transcript were detected in colon, while very high levels of both transcripts were found in skeletal muscle. Expression of mRNA transcripts for the GCS heavy subunit were likewise tissue-dependent, and did not necessarily correlate with the level of GCS1 transcripts in any tissue.

Amino Acid Sequence↗

Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori.

Fibroin light chain (L-chain) mRNA (mol. wt 4.0 X 10(5) daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development.

Animals↗

Influence of light-curing procedures and photo-initiator/co-initiator composition on the degree of conversion of light-curing resins.

OBJECTIVE: The hypothesis that the degree and rate of conversion can be modified favourably by using different light-curing procedures and different photo initiator/co-initiator combinations was tested. METHOD: A photo-initiator (0.02 mM/g resin); either camphorquinone (CQ) or 1-phenyl-1,2-propanedione (PPD), was mixed with bisGMA:TEGDMA (50:50 by weight). In addition, a co-initiator (0.04 mM/g resin); either N,N-dimethyl-p-aminobenzoic acid ethylester (DABE), N,N-cyanoethylmethylaniline (CEMA), or 2-dimethylaminoethyl methacrylate (DMAEMA), was added. These six combinations were subjected to three curing conditions (standard curing, soft-start curing or LED curing). The conversion levels (DC) were determined with differential scanning calorimetry (DSC). The DSC results were analysed using a general linear model (GLM) and Duncan's multiple range test and regular t-test. RESULTS: The fastest conversion initially was obtained by standard curing, followed by LED curing and soft-start curing. After 40 s of curing, conventional curing and soft-start curing produced a higher DC than LED curing. However, strong interactions occurred between the different variables (curing method, initiator and co-initiator). Initially, CQ was more efficient than PPD, but after 40 s, this difference was insignificant. CONCLUSION: By using soft-start curing and an appropriate photo initiator/co-initiator combination it is possible to achieve slow curing and a high DC at within a curing time of 40 s.

Acrylic Resins↗

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13,472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3'-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5'-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.

Amino Acid Sequence↗

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.

Cell Line↗

Development of a ginkgo biloba fingerprint chromatogram with UV and evaporative light scattering detection and optimization of the evaporative light scattering detector operating conditions.

A fingerprint chromatogram of a standardized Ginkgo biloba extract is developed on a monolithic silica column using a ternary gradient containing water, iso-propanol and tetrahydrofuran. For the detection, UV and evaporative light scattering (ELS) detectors are used, the latter allowing detection of the poor UV absorbing compounds as ginkgolides (A-C and J) and bilobalide in the extract. The complementary information between the UV and ELS fingerprint is evaluated. The ELS detector used in this study can operate in an impactor 'on' or 'off' mode. For each mode, the operating conditions such as the nebulizing gas flow rate, the drift tube temperature and the gain are optimized by use of three-level screening designs to obtain the best signal-to-noise (S/N) ratio in the final ELS fingerprint chromatogram. In both impactor modes, very similar S/N ratios are obtained for the nominal levels of the design. However, optimization of the operating conditions resulted, for both impactor modes, in a significant increase in S/N ratios compared to the initial evaluated conditions, obtained from the detector software.

Chromatography, High Pressure Liquid↗

Light and electron microscopic studies of lectin binding to the glycocalyx of rat pancreatic cells. II. Light microscopic changes after induction of an olive-oil pancreatitis.

Using a battery of 7 horseradish peroxidase marked lectins (WGA, RCA I, PHA, LCA, PNA, UEA I, LPA) or 2 unmarked lectins (Con A, VAA I) and HRP-marked antibodies, the binding to acinar cells with a postembedding technique on semithin sections of rat pancreatic tissue after olive-oil pancreatitis was studied light microscopically. The lectin binding of the normal healthy rat pancreatic tissue (Jonas et al. 1991) changed remarkably. Whereas the apical glycocalyx of acinar cells with the strong binding of WGA, RCA I, and PHA remained unchanged within the first 10 min of damage, the basolateral cell surface lost the typical specific binding of UEA I within the initial phase of pancreatitis just 2 min after injection of olive-oil. Con A and VAA I were found to be very reactive with the necrotic cells 60 min after administration of oil. The results were discussed in relation to the possible functions of the 2 main domains of the pancreatic acinar cell glycocalyx.

Animals↗

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Adaptation, Ocular↗

Searching for active ribosomal genes in situ: light microscopy in light of the electron beam.

Light microscopy (LM) approaches are commonly used to attain a description of the cell structure. Even though LM, if compared to electron microscopy (EM), represents a very fast approach, its resolution is, in principle, much lower than in the case of EM. To improve the LM resolution, computational methods based on removal of the image blur are frequently implemented in cell biology studies. One of the standard deblurring approaches is image restoration through deconvolution algorithms. Even though this method of mathematical remodeling of microscopically observed objects represents an efficient tool of current cell biology, it is legitimate to ask what the limits of its use are. We demonstrate that, in the specific case of the fluorescence mapping of active ribosomal genes in HeLa cell nucleoli, restoration generates a biased result. On restoration of model images, we demonstrate the difficulties of one of the most effective deconvolution algorithms during the restoration of ring-shaped fluorescent objects of a diameter comparable to the microscope resolution limit. In the case involving the mapping of nucleolar transcription in HeLa cells, not the restored fluorescence images, but rather the EM images show the true distribution of active ribosomal genes.

Cell Nucleolus↗

Amino acid sequence of rabbit light chains: variable region of a light chain from a homogeneous immunoglobulin raised by streptococcal immunization.

The variable region sequence has been determined for the light chain (L) from a rabbit homogeneous immunoglobulin (3547) produced by immunization with group A streptococcal vaccine. Unlike most immunoglobulins produced by these vaccines, this immunoglobulin had no binding activity for the group A polysaccharide nor for any of a wide range of streptococcal cell components tested, nor did it have binding activity for rabbit IgG. Tryptic digestion of the citraconylated L chain and acid hydrolysis of the aspartyl-proline bond at positions 109-110 were used to obtain two variable region peptides comprising residues 1-61 and 62-109, respectively. Automated sequence analysis of these peptides and the peptides obtained from them by complete tryptic digestion gave sequence data for the entire L-chain variable (V) region. Comparison of the 3547 L chain V region sequence with other data supports the observations that only two hypervariable regions are present in rabbit kappa chains and that the hypervariable region beginning at residue 90 may vary in length by as much as six residues.

Amino Acid Sequence↗

Simultaneous light emission from a mixture of dendrimer encapsulated chromophores: a model for single-layer multichromophoric organic light-emitting diodes.

The site isolation of two dyes capable of electronic interaction via Forster energy transfer has been studied with the two dyes coumarin 343 and pentathiophene encapsulated by dendrons containing both solubilizing and electroactive moieties. Photoluminescence studies of mixtures of the dendritic dyes show that at high dendron generation, significant site isolation is achieved with relative emission characteristics influenced by both the degree of site isolation and the emission quantum yield of the dyes. Electroluminescence studies carried out in organic light emitting diode devices confirm that color tuning may be achieved by mixing the two encapsulated dyes in a single layer. However, selective carrier trapping by one of the core component dyes can dramatically influence the effectiveness of other components in the device.

Journal Article↗

Light chain sequences of human IgM cold agglutinins (variable-region subgroups amino-acid sequence-kappa light chain-N-terminal).

The amino-terminal amino-acid sequence has been determined for the kappa light chains of nine IgM cold agglutinins with specificity for blood group-related carbohydrate antigens. Six of the nine sequences corresponded to the prototype VK(III) subgroup pattern, two to that of subgroup VK(II), and only one to the VK(I) subgroup. This distribution of kappa subgroups differs markedly from that of normal and myeloma proteins sequenced to date, where the VK(I) subgroup is more prevalent than either VK(II) or VK(III). Evidence is presented that supports the conclusion that the unusual subgroup distribution relates to the antibody property itself and not to some other attribute of these molecules.

Amino Acid Sequence↗

Characterization of a mammalian cell line that exhibits spontaneous and ultraviolet light-induced hypermutability while retaining resistance to cell killing by ultraviolet light.

Chronic exposure of V79 cells to 80 daily doses of 150 J/M2, 290-330-nm ultraviolet light (UVB) produced a mixed cell population that was found to be generally more resistant to cell killing by both UVB and UVC (254 nm) than the wild-type cells. Several subclones from this population were studied for their survival and mutation responses and then one was chosen for further characterization based on this data. The studies carried out on this subclone, designated N806, show that its spontaneous HPRT mutation rate is approximately 10 times higher than that of wild-type V79 cells and it is almost three times more mutable than the wild-type cells when both are induced by UVB or UVC. The mutation responses of N806 and MI2G cells to 50-kVp X-rays are different, but the N806 cells do not appear to be hypermutable as they are with UV. N806 cells are also moderately more resistant to the cytotoxic effects of UV radiation but are more sensitive than MI2G cells when exposed to X-rays. Assays to measure the removal of cyclobutane pyrimidine dimers (CPDs) and the incision step of nucleotide excision repair have revealed no detectable difference in the repair capacities of N806 and parental V79 cells. These results suggest that chronic, protracted UV irradiation may be able to induce a 'mutator phenotype' in a subpopulation of the progenitor cells.

Animals↗

A correlative study by electron and light microscopy of the development of type 5 adenovirus. II. Light microscopy.

The evolution of the intranuclear lesion produced by type 5 adenovirus in HEp-2 and HeLa cells is described as seen in the light microscope and the bodies formed in the course of the infection characterized histochemically. Some 12 hours after infection acidophilic protein bodies, without appreciable nucleic acid, first appear in the nucleus and coalesce into a network. Within or in association with this material, DNA-containing masses (viral aggregates) are formed which rapidly increase in amount and then coalesce. At the same time, a protein is produced, histochemically different from that of the acidophilic or basophilic structures mentioned, within the infected nucleus, which constitutes a matrix within which regular cytstals of a protein, (presumably non-viral) materialize. These structural and histochemical features are correlated with details which have been observed in parallel studies with the electron microscope.

Adenoviridae↗

Measurements from light and polarised light microscopy of human coronary arteries fixed at distending pressure.

With the long term goal of improving our understanding of the mechanisms involved in coronary artery spasm, we have undertaken a two part study of the artery structure. We have made a comparison of the relative proportions of the different layers in proximal and distal regions of the main coronary arteries, and have quantitatively assessed their three dimensional structural fabric. Major coronary arteries from nine hearts were prepared for histological examination after fixation at a transmural pressure of 120 mm Hg. Measurements from 14 proximal and distal pairs of the cross sectioned arteries showed a dominant subendothelial layer, which diminished in thickness distally, with a small fraction of muscle cells interspersed with collagen. Three dimensional orientation measurements of the collagen and muscle components, which are birefringent, were obtained from one pair of segments from each of the left anterior descending, circumflex and right coronary arteries, using the polarising light microscope and Universal stage. Findings showed (1) a single circumferential order of adventitial collagen, with a mean circular standard deviation (CSD) of 22.3 degrees; (2) very highly ordered medial smooth muscle, mean CSD of 5.0 degrees (both findings are quantitatively similar between proximal and distal segments of artery, and between arteries); and (3) a multilayered fabric of collagen in the subendothelium in all vessel segments. The principal contributor to functional differences between proximal and distal regions may be the prominent and structurally varied subendothelial layer of the coronary arteries.

Adult↗

Complex light: dynamic phase transitions of a light beam in a nonlinear nonlocal disordered medium.

The dynamics of several light filaments (spatial optical solitons) propagating in an optically nonlinear and nonlocal random medium is investigated using the paradigms of the physics of complexity. Cluster formation is interpreted as a dynamic phase transition. A connection with the random matrices approach for explaining the vibrational spectra of an ensemble of solitons is pointed out. General arguments based on a Brownian dynamics model are validated by the numerical simulation of a stochastic partial differential equation system. The results are also relevant for Bose condensed gases and plasma physics.

Journal Article↗

Light modulation of glucose-6-phosphate dehydrogenase: partial characterization of the light inactivation system and its effects on the properties of the chloroplastic and cytoplasmic forms of the enzyme.

Light inactivation of glucose-6-phosphate dehydrogenase within the pea (Pisum sativum L.) leaf chloroplast has a narrow pH optimum between 7.2 and 7.4 and is NADP-sensitive. The pH optimum for dark activation is slightly lower. Inactivation apparently results in a simple decrease in maximal velocity of the chloroplastic and cytoplasmic forms of the enzyme with no concomitant change in pH optimum or K(m) (glucose 6-phosphate).

Journal Article↗

Effect of Growth Temperature on the Lipid and Fatty Acid Composition, and the Dependence on Temperature of Light-induced Redox Reactions of Cytochrome f and of Light Energy Redistribution in the Thermophilic Blue-Green Alga Synechococcus lividus.

The thermophilic blue-green alga Synechococcus lividus was grown at 55 and 38 C. Arrhenius plots of the transient reduction of cytochrome during actinic illumination with light that excited both pigment systems revealed breaks near 43 and 26 C for cells grown at 55 C. In cells grown at 38 C these breaks occurred near 37 and 28 C, respectively. The shift from pigment state 1 to state 2 measured by fluorescence transients also showed characteristic breaks in the Arrhenius plots at 44 C for cells grown at 55 C and at 37 to 38 C and possibly at 25 C for cells grown at 38 C. The break points in the Arrhenius plots for the state shift as well as for the cytochrome f reduction are discussed in relation to phase transitions of thylakoid membrane lipids as studied by the temperature dependence of chlorophyll a fluorescence.The variations of fatty acid composition with growth temperature was also studied. When the growth temperature was lowered from 55 to 38 C, the amount of the saturated fatty acid 18:0 in the negatively charged lipids sulfoquinovosyl diglyceride and phosphatidyl glycerol decreased while the unsaturated fatty acids 18:1 and 16:1 increased. In mono- and digalactosyl diglycerides the saturated fatty acids 18:0 and 16:0 decreased and the unsaturated fatty acid 16:1 increased. In general there was an increase in the more fluid lipids in all of the lipid classes when the cells were grown at the lower temperature.

Journal Article↗