BBA - General Subjects (v.1722, #2)

Enhanced catalytic and conformational stability of Atlantic cod trypsin upon neoglycosylation by R. Venkatesh; S. Srimathi; A. Yamuna; G. Jayaraman (113-115).
The applicability of psychrophilic enzymes is limited because of their lower thermodynamic stability in spite of their higher catalytic rate. In this study, we have shown that the thermodynamic stability of the psychrophilic Atlantic cod trypsin could be enhanced appreciably by covalent chemical modification with oxidized sucrose polymer without affecting its hydrolytic activity. The acquired stability of cod trypsin was found to be on par with the mesophilic porcine trypsin.
Keywords: Atlantic cod trypsin; Psychrophilic enzyme; Neoglycosylation; Sucrose polymer; Thermodynamic stability;

Cytochrome P450 expression and catalytic activity in coronary arteries and liver of cattle by Emanuela Grasso; Vincenzo Longo; Flavio Coceani; Pier Giovanni Gervasi (116-123).
There is increasing evidence that cytochrome P450 (CYP) enzymes are involved not only in the metabolism of xenobiotics, but also in vascular homeostasis. Among the CYP-derived vasoactive agents, special importance is assigned to endogenous products from arachidonic acid (AA). Specifically, the vasodilator epoxyeicosatrienoic acids (EETs), being linked to the CYP 2B, 2C, and 2J subfamilies, and the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) connected instead to the CYP 4A subfamily and, to a lesser degree, to isoforms of the CYP 1A and 2E subfamilies. Here, we have examined the occurrence of functional CYP isoforms in the coronary arteries of cattle by RT-PCR with sequence verification, Western immunoblotting, and analysis of distinct catalytic activities with fluorescent substrate probes. Liver tissue was examined comparatively. Coronary tissue expressed mRNA transcripts and immunoreactive proteins belonging to the CYP 1A, 2C, 2E, and 2J subfamilies. Appropriate catalytic activity was ascertained with all these CYP species except 2J. A broader spectrum of CYP enzymes (CYP 1A, 2B, 2C, 2D, 2E, 2J, 3A, 4A subfamilies) was found in liver tissue with catalytic activities exceeding many fold those of coronary tissue. We conclude that bovine coronary arteries are endowed with a full-fledged CYP system with potential for AA-linked vasoregulation through dilator rather than constrictor agents. The same tissue and, to a much larger degree, liver tissue possess the capability of metabolizing xenobiotics via the CYP pathway.
Keywords: Cytochrome P450; Cattle; Coronary artery; Liver; Vasoregulation; Metabolism of xenobiotic;

Binding of hypocrellin B to human serum albumin and photo-induced interactions by Baozhong Zhao; Jie Xie; Jingquan Zhao (124-130).
Molecular binding of hypocrellins to human serum albumin (HSA) needs to be further clarified considering the phototherapeutic potentials of hypocrellins to vascular diseases. In the current work, it was estimated that the binding constant of hypocrellin B (HB) to HSA was 2.28×104 M−1. Furthermore, based on the fluorescence responses for both HB and the tryptophan of HSA, it was suggested that the binding of HB to HSA should be more specific rather than distributed randomly on the surface of HSA, which was also confirmed by photobleaching of the tryptophan via photosensitization of HB. Besides, it was found that both of the photo-bleaching of the tryptophan and the photo-oxidation of HB were principally oxygen-dependent, suggesting reactive oxygen species generated via the photosensitization of HB, instead of the free radicals of the photosensitizer (HB •− ), play the most important role in photodynamic processes.
Keywords: Hypocrellin; Human serum albumin; Specific or nonspecific binding; Photo-induced process;

Melanotransferrin (MTf) is a transferrin homologue initially identified in melanoma cells. Serum transferrin (Tf) contains two iron (Fe)-binding sites and plays a vital role in Fe transport. However, human MTf has only a single, high affinity, Fe-binding site. Furthermore, while isolated MTf can bind Fe, it plays little role in Fe uptake by cells and its function remains elusive. To further understand the biological role of this molecule, we examined the expression profile of mouse MTf (mMTf) and human MTf (hMTf) and the splice variant of the latter. Analysis of mMTf in 18 normal mouse tissues and 4 embryonic stages (7–17 days) using an RNA dot blot demonstrated it was expressed at high levels in the pancreas, salivary gland and epididymis of the adult, while embryonic tissues showed low expression. The expression pattern was very different from that of mouse transferrin receptor 1 (TfR1) mRNA, which was found at high levels in the spleen and embryo. Using the more sensitive RT-PCR technique, mMTf expression was demonstrated across all 24 normal mouse tissues assessed. Analysis of the mMTf genomic sequence predicted only one mMTf transcript, although two putative transcripts were found in the testis using Northern blotting. An alternate hMTf transcript, hΔMTf, has been identified by others, although its tissue distribution was not previously examined. In human heart and skeletal muscle, three putative hMTf transcripts were identified at approximately 2, 3 and 4 kb, the smallest transcript being consistent with hΔMTf. The two larger transcripts were also found in 10 other human tissues. The hΔMTf transcript was detected using RT-PCR and Southern blotting in tumour-derived cell lines, with the highest expression being identified in melanoma cells. Immunohistochemistry showed that hMTf was expressed primarily within epithelia. In fact, the most pronounced expression was within the epidermis of the skin, tubules of the kidney and the ducts of sweat and salivary glands. The distribution of MTf and its splice variants may provide clues to their possible biological roles.
Keywords: Transferrin; Melanotransferrin; Tumour antigen p97; Transferrin homologue;

Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells by Martin Vejražka; Radan Míček; Stanislav Štípek (143-147).
Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.
Keywords: Apocynin; NADPH-oxidase; Reactive oxygen species;

We have demonstrated previously that pretreatment of GH3 pituitary cells with muscarinic agonists may induce a higher cAMP formation in response to vasoactive intestinal peptide (VIP) or forskolin. In the present study, we further examined the adenylate cyclase (AC) that may be involved. We found that carbachol-pretreatment enhanced both VIP- and forskolin-activated AC activities. The addition of calcium ions to the incubation buffer diminished this enhancing effect. Carbachol was found to induce a decrease in intracellular calcium concentration [Ca2+]i by inhibiting calcium influx through L-type Ca2+ channels. However, the incubation of cells in Ca2+-free buffer or in the presence of L-type Ca2+ channel blockers had no influence on forskolin-stimulated cAMP formation, although both treatments induced decreases in [Ca2+]i as carbachol did. On the other hand, incubation in the presence of LaCl3 at a low concentration not being able to enter cells, forskolin-stimulated cAMP formation as well as the enhancing effect of carbachol-pretreatment on this response, were both suppressed. Similar phenomena were observed when membrane-bound AC activities were measured in the presence of LaCl3. Taken together, these results seem to suggest that pretreatment of GH3 cells with muscarinic receptor agonist may activate a Ca2+-inhibitable AC for a higher stimulated response. Low intracellular calcium concentrations are essential but not sufficient for this effect.
Keywords: cAMP; Muscarinic receptor; GH3 pituitary cell; Adenylate cyclase; Calcium;

In vivo and in vitro effect of Capsicum annum proteinase inhibitors on Helicoverpa armigera gut proteinases by Vaijayanti A. Tamhane; Nanasaheb P. Chougule; Ashok P. Giri; Anirudha R. Dixit; Mohini N. Sainani; Vidya S. Gupta (156-167).
Two proteinase inhibitors (PIs), CapA1 and CapA2, were purified from Capsicum annum Linn. Var. Phule Jyoti leaves and assessed for their in vitro and in vivo activity against Helicoverpa armigera gut proteinases (HGPs). Both the inhibitors exhibited molecular weights of about 12 kDa with inhibitory activity against bovine trypsin and chymotrypsin indicating presence of probable two-inhibitor repeats of PIN II family. CapA1 and CapA2 inhibited 60–80% HGP (azocaseinolytic) activity of fourth instar larvae feeding on various host plants while 45–65% inhibition of HGP activity of various instars (II to VI) larvae reared on artificial diet. The partial purification of HGP isoforms, their characterization with synthetic inhibitors and inhibition by C. annum PIs revealed that most of the trypsin-like activity (68–91%) of HGPs was sensitive to C. annum PIs while 39–85% chymotrypsin-like activity of HGPs was insensitive to these inhibitors. The feeding of C. annum leaf extracts and two purified PIs in various doses to H. armigera larvae for two successive generations through artificial diet demonstrated their potential in inhibiting larval growth and development, delay in pupation period and dramatic reduction in fecundity and fertility. This is the first report-demonstrating efficacy of C. annum PIs against insect gut proteinases as well as larval growth and development of H. armigera.
Keywords: Capsicum annum; Proteinase inhibitor; Helicoverpa armigera; Gut proteinase; Insect resistance;

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.
Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Subcellular localization; Protein expression; Enzyme activity;

Identification and characterization of a l-tyrosine decarboxylase in Methanocaldococcus jannaschii by Nicole D. Kezmarsky; Huimin Xu; David E. Graham; Robert H. White (175-182).
Methanofuran is the first coenzyme in the methanogenic pathway used by the archaeon Methanocaldococcus jannaschii, as well as other methanogens, to reduce CO2 to methane. The details of the pathway for the biosynthesis of methanofuran and the responsible genes have yet to be established. A clear structural element in all known methanofurans is tyramine, likely produced by the decarboxylation of l-tyrosine. We show here that the mfnA gene at M. jannaschii locus MJ0050 encodes a thermostable pyridoxal phosphate-dependent l-tyrosine decarboxylase that specifically produces tyramine. Homologs of this gene are widely distributed among euryarchaea but are not specifically related to known bacterial or plant tyrosine decarboxylases.
Keywords: l-Tyrosine decarboxylase; Coenzyme biosynthesis; Methanofuran; Methanocaldococcus jannaschii;

Analysis of collagen fibril diameter distribution in connective tissues using small-angle X-ray scattering by Kheng Lim Goh; Jennifer Hiller; J. Louise Haston; David F. Holmes; Karl E. Kadler; Ann Murdoch; Judith R. Meakin; Timothy J. Wess (183-188).
Analysis of the diameters of collagen fibrils provides insight into the structure and physical processes occurring in the tissue. This paper describes a method for analyzing the frequency distribution of the diameters of collagen fibrils from small-angle X-ray scattering (SAXS) patterns. Frequency values of fibril diameters were input into a mathematical model of the form factor to calculate the equatorial intensity which best fits the experimentally derived data from SAXS patterns. A minimization algorithm utilizing simulated annealing (SA) was used in the fitting procedure. The SA algorithm allowed for random sampling of the frequency values, and was run iteratively to build up an optimized frequency distribution of fibril diameters. Results were obtained for collagen samples from sheep spine ligaments. The mean fibril diameter value obtained from this data-fitting method was 73 nm±20 nm (S.D.). From scanning transmission electron microscopy, the mean diameter was found to be 69 nm±14 nm (S.D.). The good agreement between the two methods demonstrates the reliability of the SAXS method for the tissue examined. The non-destructive nature of this technique, as well as its statistical robusticity and capacity for large sampling, means that this method is both quick and effective.
Keywords: Collagen fibril diameter; Scanning transmission electron microscopy; Small-angle X-ray scattering; Simulated annealing;

Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system by Noriko Shinozaki-Kuwahara; Teruaki Shiroza; Mitsuo Hayakawa; Yoshimitsu Abiko; Kazuo Fukushima (189-199).
We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5′-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5′-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration.The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.
Keywords: Glucosyltransferase; Streptococcus sobrinus; Streptococcus anginosus; Integration; Biofilm;

Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence by Takuya Koseki; Kenji Takahashi; Shinya Fushinobu; Haruyuki Iefuji; Kimio Iwano; Katsumi Hashizume; Hiroshi Matsuzawa (200-208).
We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward α-naphthylbutyrate (C4) and α-naphthylcaprylate (C8), respectively, was due to a lower K m value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K m and considerably increased k cat. The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0–8.0, 7.0, and 6.0, respectively.
Keywords: Recombinant feruloyl esterase; Flap; Site-directed mutagenesis; Substrate discrimination; pH dependence;

Oxidative damage of plasma proteins and lipids in epidemic dropsy patients: Alterations in antioxidant status by Mukul Das; Kishore Babu; Naveen P. Reddy; Lalit M. Srivastava (209-217).
Epidemic dropsy is an acute food adulterant disease caused due to consumption of edible mustard oil contaminated with argemone oil. Our in vitro studies have shown that the toxicity of argemone oil is due to the production of reactive oxygen species. The present study was aimed to evaluate the development of oxidative stress in terms of oxidation of plasma proteins and lipids and its correlation to enzymatic and non-enzymatic antioxidants in epidemic dropsy patients. Total plasma protein and globulin contents were found to be significantly (P<0.05) enhanced with a concomitant decrease (P<0.05) in albumin/globulin ratio in dropsy patients when compared to controls. Total cholesterol, triglycerides, low density lipoprotein cholesterol and very low density lipoprotein cholesterol were found to be significantly (P<0.05) increased with a simultaneous decrease (51%) in high density lipoprotein cholesterol in dropsy patients. The oxidation of plasma proteins and lipids were substantially enhanced (162–175%) in dropsy patients when compared to controls. Further, significant (P<0.05) decrease in superoxide dismutase, catalase, glutathione reductase and glutathione-s-transferase with a concomitant increase (69%) in glutathione peroxidase activity was noticed in dropsy patients. A significant reduction in plasma total antioxidant capacity, α-tocopherol, glutathione, retinol and retinyl esters content was observed in dropsy patients when compared to healthy controls. The results suggest that there exists an unproportionate equilibrium between free radicals formation and enzymatic and non-enzymatic antioxidant scavengers, which may cause oxidative damage to proteins and lipids in dropsy patients.
Keywords: Epidemic dropsy; Argemone oil; Free radical; Oxidative stress; Lipid peroxidation; Protein carbonyl group; Total antioxidant capacity; Antioxidant enzyme and non-enzymatic antioxidant;

The intestinal absorption of benzyl β-glucoside (BNZβglc) contained in the fruit of Prunus mume SIEB. et ZUCC. (Rosaceae), which is traditionally used as a medicinal food in Japan, was studied in rat intestines. BNZβglc was absorbed from the mucosal to serosal sides. Its metabolite, benzyl alcohol (BAL), was also detected on both the mucosal and serosal sides. In the presence of phloridzin (Na+/glucose cotransporter (SGLT1) inhibitor) or in the absence of Na+ (driving force), BNZβglc absorption was significantly decreased. Transport clearance of BNZβglc across the brush border membrane decreased as its concentration increased. These results indicate that BNZβglc is transported by SGLT1. Metabolic clearance of BNZβglc also decreased as its concentration increased. The amount ratio of BNZβglc to BAL on the serosal side increased with the increase of BNZβglc concentration. The intestinal availability of BNZβglc was lower in the absence of Na+ than in the presence of Na+, indicating that the SGLT1-mediated transport of BNZβglc increases intestinal availability by decreasing the intestinal extraction ratio. This neutraceutical study concluded that intestinal carrier-mediated transport across the brush border membrane improves the intestinal availability of nutritionally, pharmacologically or physiologically active compounds that undergo intestinal metabolism (first-pass effect).
Keywords: Benzyl glucoside; Intestinal absorption; Intestinal availability; SGLT1; Prunus mume;

Biologically active bisquaternary ammonium chlorides: physico–chemical properties of long chain amphiphiles and their evaluation as non-viral vectors for gene delivery by Emilia Fisicaro; Carlotta Compari; Elenia Duce; Gaetano Donofrio; Bożenna Różycka-Roszak; Edyta Woźniak (224-233).
The biological properties of bisquaternary ammonium salts, which are derivatives of N,N-bisdimethyl-1,2-ethanediamine (bis-C n BEC), of general formula /C n H2n+1OOCCH2(CH3)2N+CH2CH2N+(CH3)2CH2COOC n H2n+1/2Cl, were investigated (n=10, 12, 14). The interaction with model membrane was studied by differential scanning calorimetry experiments, and the apparent adiabatic molar compressibility of their solution as a function of concentration was obtained by sound velocity measurements. Their biological activities were assayed by Electrophoresis Mobility Shift, MTT proliferation, and transient transfection. All the investigated compounds interact with the DNA and are able to transfect DNA, when they are coformulated with DOPE, with an efficiency significantly greater than that of a standard commercial transfection reagent. Bis-C14BEC is the only molecule able to deliver DNA inside the cells without a helper lipid, as shown by EGFP expression, albeit with a low efficiency in comparison with a standard commercial transfection reagent. This may be due to a slightly different interaction of bis-C14BEC from bis-C10BEC and bis-C12BEC with phospholipid bilayers. Bis-C10BEC and bis-C12BEC show a slight fluidising effect, while bis-C14BEC increases stability of both the gel and the rippled gel phases.
Keywords: Bisquaternary ammonium salt; DPPC liposome; Non-viral vector for gene delivery; Differential scanning calorimetry; Sound velocity measurement; Apparent adiabatic molar compressibility; Electrophoresis mobility shift assay; Transfection; Green fluorescent protein;