Functional inactivation by interleukin-1β of glyceraldehyde-3-phosphate dehydrogenase in insulin-secreting cells

Aims/hypothesis: It is well established that long-term exposure of isolated cells to cytokines [e.g., IL-1] results in increased expression of inducible nitric oxide synthase and subsequent release of nitric oxide, which in turn, has been shown to mediate a wide array of effects, including alterations in cellular high-energy metabolism. In this context, several extant studies have demonstrated significant reduction in adenine and guanine nucleotide triphosphate levels in cells exposed to IL-1. Herein, we examined the functional status of glyceraldehyde-3-phosphate dehydrogenase [GAPDH] in insulin-secreting cells exposed to IL-1, since it represents the first enzyme in the glycolytic pathway that is involved in the generation of ATP. Methods: GAPDH was assayed spectrophotometrically in the cytosolic fraction derived from control and IL-1 -treated [300 pM for 24 hrs] insulin-secreting cell lines [HIT-T15 and RINm5F]. Results: IL-treatment resulted in marked attenuation of GAPDH activity in HIT and RIN cells; such a reduction in this activity was not due to inhibition of its expression by IL-1. Instead, we observed that incubation of HIT and RIN lysates with peroxynitrite, a reactive intermediate of nitric oxide with superoxide anion, resulted in significant reduction in the GAPDH activity. Conclusion/interpretation: These results identify a GAPDH as one of the biochemical loci for the effects of IL-derived peroxynitrite in the islet cell. The previously reported reduction in high-energy phosphate levels in an IL-treated cell may, in part, be due to inhibition of GAPDH activity, and subsequent reduction in the glycolytic efficiency of the cell.     

Prediction of the Secondary Structure Contents of Globular Proteins Based on Three Structural Classes

The prediction of the secondary structural contents (those of -helix and -strand) of a globular protein is of great use in the prediction of protein structure. In this paper, a new prediction algorithm has been proposed based on Chou's database [Chou (1995), Proteins 21, 319–344]. The new algorithm is an improved multiple linear regression method, taking into account the nonlinear and coupling terms of the frequencies of different amino acids and the length of the protein. The prediction is also based on the structural classes of proteins, but instead of four classes, only three classes are considered, the class, class, and the mixed + and / class or simply the class. Thus the ambiguity that usually occurs between + proteins and / proteins is eliminated. A resubstitution examination for the algorithm shows that the average absolute errors are 0.040 and 0.035 for the prediction of -helix content and -strand content, respectively. An examination of cross-validation, the jackknife analysis, shows that the average absolute errors are 0.051 and 0.045 for the prediction of -helix content and -strand content, respectively. Both examinations indicate the self-consistency and the extrapolating effectiveness of the new algorithm. Compared with other methods, ours has the merits of simplicity and convenience for use, as well as high prediction accuracy. By incorporating the prediction of the structural classes, the only input of our method is the amino acid composition and the length of the protein to be predicted.     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

Production of glucosamine containing disaccharides of the Lewis-A and -X types employing glycosidases

Disaccharide derivatives of interest for inhibition studies and for synthesis of the blood group determinants Lewis-a and Lewis-x were obtained with glycosidases as catalysts. Thus, Fuc(1–4)(6-OBn)GlcNH₂SEt and Gal1–3(6-OBn)GlcNH₂-SEt were produced employing (6-OBn)GlcNH₂SEt as acceptor and -L-fucosidase and -D-galactosidase, respectively, as catalysts. The phthalimido derivative of lactosamine, Gal1-4GlcNPhthSEt, was prepared from lactose employing GlcNPhthSEt as the acceptor and a yeast -galactosidase as catalyst. The reactions were both regio- and stereospecific, which allowed straightforward production of pure products on a g scale and higher.     

Bcl-2 blocks apoptotic signal of transforming growth factor-β in human hepatoma cells

Transforming growth factor- (TGF-) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF- induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF- in three well differentiated human hepatoma cell lines. Two TGF- sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF- resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-X_L, Bcl-X_S and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF--induced apoptosis. TGF- treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF--F-induced apoptosis by interfering TGF- generated signals leading to induce reactive oxygen species production.     

Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: ¹KG(R)V⁵KGTGD¹⁰(T)PXXV¹⁵XLY(N)R²⁰T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man2Man6(Man3) Man6(Man2Man2Man3)Man4GlcNAc4GlcNAc (13%); ± Man2Man6(Man3)Man6(± Man2 Man2 Man3)Man4GlcNAc4GlcNAc (29%); Man6(Man3) Man6(Man2Man3)Man4GlcNAc4GlcNAc (13%); Man6(Man3)Man6(Man3)Man4GlcNAc4GlcNAc (16%); Man6(Man3)(Xyl2)Man4GlcNAc 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.Abbreviations ConA concanavalin A - Endo H endo-N-acetyl--d-glucosaminidase H - GlcNAc N-acetylglucosamine - gu glucose unit - IEF isoelectric focusing - Man mannose - NEPHGE non-equilibrium pH gradient electrophoresis - PNGase F peptide-N-glycosidase F - PVDF polyvinylidenedifluoride - Xyl xylose We thank Geoffrey Guile (Oxford Glycobiology Institute, Oxford, UK) for help with HPLC separations and amino acid and hexosamine analysis, Terry Butters (Oxford Glycobiology Institute) for providing the exoglycosidases and advice in their use, Manuela Regala (Instituto de Tecnologia Química e Biológica Oeiras, Portugal) and Paula Veríssimo (University of Coimbra, Portugal) for determining the N-terminal amino acid sequence of G20 and G23 and Dr. Jorge Lampreia (Universidade Nova de Lisboa, Lisbon, Portugal) for the computerised search of the SWISS-PROT data bank. Lupinus albus seeds were provided by Dr. João Neves Martins (Instituto Superior de Agronomia Lisbon, Portugal). We also thank J. Romão (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for technical assistance in antibody production. This work was supported by Junta National de Investigação Científica e Tecnológica, Portugal.     

Ecto-ATPase/phosphatase activity in the olfactory sensilla of the spiny lobster,Panulirus argus: localization and characterization

Summary Electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has chemoreceptors that are selectively excited by adenine nucleotides in seawater. Biochemical studies have revealed that these same nucleotides can be rapidly dephosphorylated by ectoenzymes associated with the olfactory sensilla (aesthetascs). In this study the distribution of ecto-ATPase/phosphatase activity within aesthetascs was determined cytochemically and the nature of the adenine-nucleotide dephosphorylating activity was dissected biochemically. Cytochemically, the distribution of ATP-dephosphorylating activity was similar to that shown previously for AMP and -glycerol phosphate; i.e., cerium phosphate reaction product was specifically localized to the transitional zone where the sensory dendrites develop cilia and branch to form the outer dendritic segments. Unlike the dephosphorylation of AMP and -glycerol phosphate, Mg²⁺ or Ca²⁺ was required for ecto-ATPase/phosphatase activity. Biochemical measures of both AMP-and ATP-dephosphorylating activity within aesthetascs corroborated the cytochemical evidence that these activities are localized to the transitional zone. A major portion of the AMP dephosphorylation (about 67%) derives from nonspecific alkaline phosphatase activity that is insensitive to levamisole and L-bromotetramisole. In contrast, nonspecific phosphatase activity accounted for a much smaller part of the ATP dephosphorylation (about 15%). Ectoenzymatic activity in the transitional zone may be an important means of removing excitatory/inhibitory nucleotides from this region.Abbreviations ADP Adenosine 5-diphosphate - AMP adenosine 5-monophosphate - AMPCP , -methylene ADP - ASW artificial seawater - ATP adenosine 5-triphosphate - -GP -glycerol phosphate - EM electron microscopy     

Glycosylation patterns of membrane proteins of the jellyfish Cyanea capillata

Integral and membrane-associated proteins extracted from neuron-enriched perirhopalial tissue of the jellyfish Cyanea capillata were probed with a panel of lectins that recognize sugar epitopes of varying complexity. Of the 13 lectins tested, only concanavalin A, jacalin lectin and tomato lectin stained distinct bands on Western blots, indicating the presence of repeating -1,6-mannoses, terminal Gal--1,6-GalNAc and repeating -1,4-linked GlcNAc, respectively. In whole-mounted perirhopalial tissue, jacalin lectin stained several cell types, including neurons, muscle, cilia and mucus strands. Tomato lectin stained secretory cells intensely, and neurons in a punctate fashion. Concanavalin A stained cytoplasmic epitopes in both ecto-and endodermal cells, and ectodermal secretory cells and the mucus strands emanating from them. With the exception of tomato lectin's sugar epitope, the other sugar epitopes identified in this study are non-complex. This study suggests that while glycosylation of integral and membrane-associated proteins occurs in Cyanea, the sugars post-translationally linked to these proteins tend to be simple.     

Water stress-induced oxidative damage and antioxidant responses in micropropagated banana plantlets

Oxidative injury and antioxidant responses were investigated in two banana genotypes (Musa AAA Berangan and Musa AA Mas) subjected to 40 % PEG-induced water stress. PEG treatment resulted in oxidative injury, as expressed in increased lipid peroxidation and reduced membrane stability index, in both cultivars; however, greater oxidative injury was detected in Mas. Under PEG treatment, catalase activity and glutathione reductase activity were enhanced in both cultivars, but were higher in Mas. Ascorbate peroxidase activity was enhanced in Berangan under water stress, but was unaffected in Mas. Meanwhile, superoxide dismutase activity was inhibited in both cultivars under water stress, but higher activity was detected in Berangan. Higher ascorbate peroxidase and superoxide dismutase activities were associated with greater protection against water stress-induced oxidative injury.     

Characterization of T cell hybridomas raised against a glycopeptide containing the tumor-associated T antigen, (betaGal (1-3) alphaGalNAc-O/Ser)

T cell hybridomas were raised against the glycopeptide S⁷² (Core-1) containing the tumor-associated disaccharide Gal (1–3) GalNAc (Core-1) O-linked to serine at position 72 in the mouse hemoglobin derived decapeptide Hb (67–76). All hybridomas recognized the glycopeptide S⁷² (Core-1). Two of the selected hybridomas responded, however, much better to the S⁷² (Tn) glycopeptide containing the monosaccharide GalNAc O-linked to serine. In addition, one hybridoma cross-responded to the glycopeptide T⁷² (Core-1) having a threonine at position 72 instead of a serine. No cross-responses were found to other glycopeptides consisting of the same hemoglobin peptide with different glycans attached or to the unglycosylated peptides. The T cell receptor V and V usage was clearly diverse. The CDR3 regions demonstrated moreover a predominance of small polar amino acid side chains, and three hybridomas contained a common sequence motif. All the sequenced CDR3 regions contained furthermore a conserved proline-glycine motif. In conclusion, immunization with the disaccharide containing glycopeptides S⁷² (Core-1) created a heterogeneous population of glycopeptide specific T cells with the ability of cross-responding toward related glycopeptides.     

Temporal relationship between immune cell influx and the expression of inducible nitric oxide synthase, interleukin-4 and interferon-gamma in pancreatic islets of NOD mice following adoptive transfer of diabetic spleen cells

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon- and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p=0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon- was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon- staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon- and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.     

The ultrastructure of the cell types in the endocrine pancreas of the horse

Summary The islets of Langerhans of the equine pancreas were examined with the electron microscope after immersion or perfusion fixation. Five cell types could be distinguished after fixation by either technique: 1. A-cells, situated at the center of the islets, 2. B-cells, containing mostly pale granules and constituting the principal cell type of the periphery of the islets, 3. D-cells, also located mainly at the periphery of the islets, 4. G-cells, found at the edge of the islets and in the exocrine pancreas, and 5. S-cells, (small granule cells), which are relatively few in number and occur only in the islets. The function and age-dependent modifications of these cells are discussed. The formation of light and dark cells and of mixed cells are regarded as artifact, since cells of this type occur only under the condition of immersion fixation.Dedicated to Professor Dr. Drs. h.c. W. Bargmann on the occasion of his 70th birthday.     

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-α through IL-12/TNF-α/NF-κB autocrine loop

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor (TNF-). Mo-DCs-Tum showed higher secretions of IL-12 and TNF- than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-B) activation was also induced in Mo-DCs-Tum within 6 h. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF- secretions from Mo-DCs-Tum. Administration of recombinant TNF- or IL-12 enhanced IL-12 or TNF- secretion respectively in Mo-DCs-Tum. Addition of anti-TNF- or anti-IL-12 neutralizing antibody decreased NF-B activation and IL-12 or TNF- secretion in Mo-DCs-Tum. These results suggest that TNF- or IL-12 secretion induces NF-B activation, and it stimulates further TNF- and IL-12 secretions, i.e., an IL-12/TNF-/NF-B autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF- through accelerated NF-B activation induced by the IL-12/TNF-/NF-B autocrine loop.     

Laminins, tenascin and type VII collagen in colorectal mucosa

Summary The distribution of different laminin polypeptides, type VII collagen and tenascin has been studied in adult and foetal colorectal mucosa by using the indirect immunofluorescence technique. Immunoreactivity for laminin 1 chain was located to basement membranes of epithelia, muscularis mucosae, and blood vessels, respectively in different segments of adult colon and rectum. Laminin 1 and 1 chains were additionally expressed in lamina propria. Laminin 2 chain was also found in lamina propria around the pericyptal fibrollasts. Immunoreactivity for laminin 2 chain was restricted to basement membranes in the muscularis mucosae and arteries. Laminin 3 and 3 chains, suggestive for laminin-5, were confined especially to surface epithelial basement membranes. Immunoreactivity for type VII collagen was confined to basement membrane of surface epithelium in a punctate manner, while that for tenascin was seen slightly more broadly in the basement membrane zone and also in the muscular layer. The distribution of laminin chains in 16-week-foetal colon mostly resembled that of corresponding adult tissue, although immunoreactivities for laminin 2 and 2 chains were lacking. Type VII collagen and the high molecular weight isoform of tenascin also absent from the foetal colon. The results show that the basement membrane of the surface epithelium of colon and rectum express the components of epithelial adhesion complex, laminin-5 (3-3-2) and type VII collagen, resembling in this respect small intestine and stomach while laminin-2 (2-1-1) appears to be associated with pericryptal fibroblasts, and laminin-1 (1-1-1) widely in most basement membranes.     

GM1 Inhibits Amyloid β-Protein-Induced Cytokine Release

The ganglioside GM1 is known to play a pivotal role in neuronal survival and/or regeneration. Recently it has been shown that GM1 binds tightly with membrane-bound amyloid protein (A) and prevents its conversion from a helical to a -sheet structure. To examine the potential physiological consequences of this binding, we studied the effect of GM1 on A-stimulated release of proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and TNF-, using the human monocytic cell line, THP-1, as a model system. Treatment of THP-1 cells with A 1–40 or A 25–35 resulted in an increased cytokine release from these cells. However, treatment of A-activated THP-1 cells with GM1 and several other complex gangliosides, but not hematosides and neutral glycosphingolipids such as asialo-GM1 (GA1), lactosylceramide, and globoside, significantly decreased the cytokine release. In contrast, this effect was not observed for lipopolysaccharide (LPS)-activated and thrombin-activated THP-1 cells, indicating that the ganglioside effect is specific for A-induced cytokine release. A direct interaction between GM1 and A was demonstrated using the surface plasmon resonance technique. We found that GM1 ganglioside exhibited higher affinity for A 1–40 than GA1, suggesting that the sialic acid moiety of GM1 is necessary for its interaction with A. We conclude that the inhibitory effect of GM1 on A-induced cytokine release may reflect pre-existing abnormalities in membrane transport at the stage of amyloid formation and that GM1 may induce conformational changes in A, resulting in diminished fibrillogenesis and prevention of the inflammatory response of neuronal cells in Alzheimer's disease.     

Peroxynitrite reactivity with amino acids and proteins

Summary. Peroxynitrite, the product of the fast reaction between nitric oxide (NO) and superoxide O₂ ^– radicals, is an oxidizing and nitrating agent which is able to traverse biological membranes. The reaction of peroxynitrite with proteins occurs through three possible pathways. First, peroxynitrite reacts directly with cysteine, methionine and tryptophan residues. Second, peroxynitrite reacts fast with transition metal centers and selenium-containing amino acids. Third, secondary free radicals arising from peroxynitrite homolysis such as hydroxyl and nitrogen dioxide, and the carbonate radical formed in the presence of carbon dioxide, react with protein moieties too. Nitration of tyrosine residues is being recognized as a marker of the contribution of nitric oxide to oxidative damage. Peroxynitrite-dependent tyrosine nitration is likely to occur through the initial reaction of peroxynitrite with carbon dioxide or metal centers leading to secondary nitrating species. The preferential protein targets of peroxynitrite and the role of proteins in peroxynitrite detoxifying pathways are discussed.     

A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.     

Comparative Study of Induction of iNOS mRNA Expression in Vascular Cells of Different Species

To determine the difference in induction of inducible nitric oxide synthase (iNOS) mRNA expression in cultured vascular cells of different species, the expression of iNOS genes and their regulatory mechanisms in rat, human, bovine, and rabbit vascular endothelial cells and smooth muscle cells (SMC) were studied by Northern blotting, chloramphenicol acetyltransferase (CAT) assay, and electrophoretic mobility shift assay (EMSA). Qualitative estimation of iNOS mRNA by Northern-blot analysis demonstrated that the combination of interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and lipopolysaccharides (LPS) drastically induces iNOS expression in rat and human SMC, and a more moderate effect was observed for endothelial cells; the effect of IL-1 alone was much weaker than that of the three factors. IL-1 alone or a mixture of IL-1, TNF-, and LPS both showed negligible effect on iNOS expression in bovine and rabbit vascular endothelial cells and SMC. Results of CAT assay corresponded well with Northern analysis indicating 7-fold increase in CAT activity by the mixture of IL-1, TNF-, and LPS in SMC and more moderate, 2-fold increase, in endothelial cells. IL-1 alone produced an intermediate effect (less than 2-fold) on vascular SMC of rats and humans. The results of EMSA showed that two shifted bands appeared when the nuclear protein from rat and human vascular endothelial cells bound to the region from –1037 to –787 of the rat iNOS gene, while vascular SMC nuclear protein only produced a single shifted band under the same conditions. These results suggest that cell- and species-specific mechanisms exist in the induction of iNOS expression.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.