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BBA - General Subjects (v.1770, #5)

Editorial Board (pp. ii).

Suprasternal gland secretion of male short-tailed opossum induces IP₃ generation in the vomeronasal organ by Dalton Wang; Ping Chen; Wei Quan; Mimi Halpern (pp. 725-732).

Chemical communication is an important component of mammalian social behaviors. Gray short-tailed opossums ( Monodelphis domestica) communicate by scent marking. The male opossum possesses a prominent suprasternal scent gland, extracts of which strongly attract female opossums. This attractivity remains unaltered following repeated lyophilization. The suprasternal gland secretion functions in a sexually dimorphic manner, i.e., it elicits elevated levels of IP₃ in the vomeronasal (VN) sensory epithelium of female opossums, but suppressed the levels of IP₃ in the VN sensory epithelium of male opossums. The elevated levels of IP₃ induced by suprasternal gland secretion in female vomeronasal sensory epithelium is inhibited by the G_i/o specific inhibitor, NF023, but not its inactive analogue, NF007. It is also suppressed by specific antibodies to the alpha subunits of G_i and G_o proteins, by the phospholipase C inhibitor, U73122, as well as by GDPβS. Surprisingly, GDPβS itself enhances basal levels of IP₃ in female VN sensory epithelium. This GDPβS-induced increase in levels of IP₃ is reduced by the PLC inhibitor, U73122, but not by the G_i/o inhibitor, NF023. In addition, GDP also enhances basal levels of IP₃. GDPβS, a known inhibitor of G-protein activation, thus appears to have dual functions: as both stimulator and inhibitor of IP₃ production in the VN sensory epithelium of opossums. In contrast, this nucleotide analogue functions as an inhibitor in the VN sensory epithelium of mice. The mechanism of signal transduction underlying the suprasternal gland secretion-elicited signals in the VN sensory epithelium of opossums appears to involve signals that are generated through activation of G-protein-coupled receptors and transduced via activation of G_i/o-proteins and the effector, phospholipase C, resulting in an increased production of the second messenger, IP₃. The extracellular signals are thus amplified.

Keywords: Suprasternal scent gland; Gland secretion; Short-tailed opossum; IP; ₃; generation; Signal transduction

A versatile equation to describe reversible enzyme inhibition and activation kinetics: Modeling β-galactosidase and butyrylcholinesterase by Ryan Walsh; Earl Martin; Sultan Darvesh (pp. 733-746).

Current treatments for Alzheimer's disease involve inhibiting cholinesterases. Conversely, cholinesterase stimulation may be deleterious. Homocysteine is a known risk factor for Alzheimer's and vascular diseases and its active metabolite, homocysteine thiolactone, stimulates butyrylcholinesterase. Considering the opposing effects on butyrylcholinesterase of homocysteine thiolactone and cholinesterase inhibitors, understanding how these molecules alter this enzyme may provide new insights in the management of dementia. Butyrylcholinesterase does not strictly adhere to Michaelis–Menten parameters since, at higher substrate concentrations, enzyme activation occurs. The substrate activation equation for butyrylcholinesterase does not describe the effects of inhibitors or non-substrate activators. To address this, global data fitting was used to generate a flexible equation based on Michaelis–Menten principles. This methodology was first tested to model complexities encountered in inhibition by imidazole of β-galactosidase, an enzyme that obeys Michaelis–Menten kinetics. The resulting equation was sufficiently flexible to permit expansion for modeling activation or inhibition of butyrylcholinesterase, while accounting for substrate activation of this enzyme. This versatile equation suggests that both the inhibitor and non-substrate activator examined here have little effect on the substrate-activated form of butyrylcholinesterase. Given that butyrylcholinesterase inhibition can antagonize stimulation of this enzyme by homocysteine thiolactone, cholinesterase inhibition may have a role in treating Alzheimer and vascular diseases related to hyperhomocysteinemia.

Keywords: Alzheimer's disease; Vascular disease; Enzyme kinetics; Equation modeling; Global data fitting; Homocysteine thiolactone; Galantamine

Enhanced cellular delivery and transfection efficiency of plasmid DNA using positively charged biocompatible colloidal gold nanoparticles by Sang Myoung Noh; Won-Ki Kim; Sun Jae Kim; Jung Mogg Kim; Kwang-Hyun Baek; Yu-Kyoung Oh (pp. 747-752).

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 (pVAXmIL-2) was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and the plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights, such as PEI25K (m.w. 25 kd) and PEI2K (m.w. 2 kd). PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than was seen with PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than was seen in association with the PEI25K/DNA and PEI2K/DNA complexes, respectively. Following intramuscular administration, PGN/DNA complexes showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. Moreover, the PGN/DNA complexes showed higher cell viability than other cationic nonviral vectors. Collectively, the results of this study suggest that the PGN/DNA complexes may harbor the potential for development into efficient and safe gene delivery vehicles.

Keywords: Nonviral gene delivery; Colloidal gold nanoparticle; Transfection efficiency; Cationic vector

Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1 by Paolo Paoli; Alessandra Modesti; Francesca Magherini; Tania Gamberi; Anna Caselli; Giampaolo Manao; Giovanni Raugei; Guido Camici; Giampietro Ramponi (pp. 753-762).

We mutated Trp¹³⁴ and Tyr¹³⁵ of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp¹³⁴ to Tyr or Ala, and Tyr¹³⁵ to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step ( k₃). Furthermore, we noted that the Trp¹³⁴ to Ala mutation causes a dramatic drop in k_cat/ K_m and a slight enhancement of the dissociation constant K_s. The conservative mutant W134Y shows a k_cat/ K_m very close to that of wild type, probably compensating the two-fold decrease of k₃ with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp¹³⁴ with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp¹³⁴ to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step.

Keywords: Abbreviations; PTP; protein tyrosine phosphatase; PTK; protein tyrosine kinase; LMW; low molecular weight; Ltp1; S. cerevisiae; LMW-PTP; p; NPP; p; -nitrophenyl phosphate; GST; glutathione S-transferase; CD; circular dichroismPTP; LMW-PTP; Ltp1; Tyrosine phosphorylation; Yeast; Mutagenesis; Aromatic residue

Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942 by Eiji Suzuki; Kazuhiro Umeda; Satoko Nihei; Katsuya Moriya; Hajime Ohkawa; Shoko Fujiwara; Mikio Tsuzuki; Yasunori Nakamura (pp. 763-773).

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light–dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.

Keywords: Cyanobacteria; Debranching enzyme; GlgX; Glycogen; Polysaccharide metabolism; Synechococcus

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity by Wioletta Górka-Nieć; Renata Bańkowska; Grażyna Palamarczyk; Hubert Krotkiewski; Joanna S. Kruszewska (pp. 774-780).

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Δ and pmt2Δ mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.

Keywords: O-mannosylation; GDP-mannose level; cis; -prenyltransferase activity; pmt; mutants

Crosslinking of the NER damage recognition proteins XPC-HR23B, XPA and RPA to photoreactive probes that mimic DNA damages by Ekaterina A. Maltseva; Nadejda I. Rechkunova; Ludovic C. Gillet; Irina O. Petruseva; Orlando D. Schärer; Olga I. Lavrik (pp. 781-789).

A new assay to probe the mechanism of mammalian nucleotide excision repair (NER) was developed. Photoreactive arylazido analogues of dNMP in DNA were shown to be substrates for the human NER system. Oligonucleotides carrying photoreactive “damages” were prepared using the multi-stage protocol including one-nucleotide gap filling by DNA polymerase β using photoreactive dCTP or dUTP analogues followed by ligation of the resulting nick. Photoreactive 60-mers were annealed with single-stranded pBluescript II SK (+) and subsequently primer extension reactions were performed. Incubation of HeLa extracts with the plasmids containing photoreactive moieties resulted in an excision pattern typical of NER. DNA duplexes containing photoreactive analogues were used to analyze the interaction of XPC-HR23B, RPA, and XPA with damaged DNA using the photocrosslinking assay. Crosslinking of the XPC-HR23B complex with photoreactive 60-mers resulted in modification of its XPC subunit. RPA crosslinked to ssDNA or mismatched dsDNA more efficiently than to dsDNA, whereas XPA did not show a preference for any of the DNA species. XPC and XPA photocrosslinking to DNA decreased in the presence of Mg²⁺ whereas RPA crosslinking to DNA was not sensitive to this cofactor. Our data establish a photocrosslinking assay for the investigation of the damage recognition step in human nucleotide excision repair.

Keywords: Nucleotide excision repair; XPC-HR23B; XPA; RPA; Photoaffinity modification

A novel ornithine-containing tripeptide isolated from the extract of the brackish-water bivalve Corbicula japonica by Hidemitsu Uchisawa; Tetsushi Naraoka; Tomotada Ono (pp. 790-796).

Previous studies have demonstrated that frozen preparations of the brackish-water bivalve Corbicula japonica significantly increase the content of free ornithine found in its extracts. Here we report a novel ornithine-containing tripeptide commonly found in C. japonica, which is believed to be the source of increased free ornithine. The new peptide, named acorbine, was isolated from extracts of this bivalve obtained using ultra-filtration and gel permeation chromatography. Acorbine is comprised of N²-[ N²-(β-alanyl)-l-ornithyl]-l-ornithine as determined by amino acid composition analysis, N- and C-terminal amino acid analyses, proton nuclear magnetic resonance spectrometry, and chirality analysis of the ornithine residue. The total amount of β-alanine and ornithine in the extract remained constant regardless of the temperature at which the bivalve was processed. The amount of free β-alanine and ornithine increased significantly when the bivalve was frozen, with a corresponding decrease in peptidic β-alanine and ornithine. The results suggest that changing the growth conditions triggers tripeptide proteolysis within the bivalve, which ultimately manifests in increased free β-alanine and ornithine.

Keywords: Ornithine; β-alanine; Peptide; Brackish-water bivalve; Corbicula japonica

The drug binding site of human α₁-acid glycoprotein: Insight from induced circular dichroism and electronic absorption spectra by Ferenc Zsila; Yasunori Iwao (pp. 797-809).

Human α₁-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug–AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via π–π stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the β-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.

Keywords: Abbreviations; AGP; human serum α; ₁; -acid glycoprotein; CD; circular dichroism; CE; Cotton effect; CHLP; chlorpromazine; CRB; carbamazepine; DPD; dipyridamole; ICD; induced circular dichroism; L/P; ligand/protein molar ratio; MFQ; mefloquine; PRQ; primaquine; PRP; propranolol; RHB; rhodamine B; TRZ; terazosine; UV/Vis; ultraviolet-visibleβ-Barrel; Human serum α; ₁; -acid glycoprotein; Ligand binding; Lipocalin; Induced circular dichroism; Non-degenerate exciton coupling; Tryptophan mutants

Purification and characterization of patagonfibrase, a metalloproteinase showing α-fibrinogenolytic and hemorrhagic activities, from Philodryas patagoniensis snake venom by M.E. Peichoto; P. Teibler; S.P. Mackessy; L. Leiva; O. Acosta; L.R.C. Gonçalves; A.M. Tanaka-Azevedo; M.L. Santoro (pp. 810-819).

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aα-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca²⁺ and inhibited by Zn²⁺, cysteine, dithiothreitol and Na₂EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.

Keywords: Colubrid snake venom; Fibrinogen; Hemorrhage; Metalloproteinase; Coagulation; Platelets

Upregulation of PHLDA2 in Dicer knockdown HEK293 cells by Kai-Fu Tang; Yan Wang; Pengfei Wang; Min Chen; Yao Chen; Huai-Dong Hu; Peng Hu; Bo Wang; Wenjie Yang; Hong Ren (pp. 820-825).

It has been reported that RNAi-dependent chromatin silencing in vertebrates is not restricted to the centromeres. To address whether RNAi machinery could regulate the chromatin structure of imprinted genes, we knocked down Dicer in HEK293 cells and found that the expression of PHLDA2, one of the several genes in the imprinted gene domain of 11p15.5, was specifically upregulated. This was accompanied by a shift towards more activated chromatin at PHLDA2 locus as indicated by change in H3K9 acetylation, however, the methylation state at this locus was not affected. Furthermore, we found that PHLDA2 was downregulated in growth-arrested HEK293 cells induced by either serum deprivation or contact inhibition. This suggests that PHLDA2 upregulation might be a direct result of Dicer depletion rather than the consequence of growth arrest induced by Dicer knockdown. Considering the reports that there is consistent placental outgrowth in PHLDA2 knockout mice and that PHLDA2 overexpression in mice causes growth inhibition, we speculate that PHLDA2 may be a candidate for contributing to the reduced growth rate of Dicer-deficient cells and the very early embryonic lethality in Dicer knockout mice.

Keywords: PHLDA2; Dicer; Genomic imprinting; Embryonic lethality; Growth arrest

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease by Irina N. Shalova; Katerina Cechalova; Zuzana Rehakova; Petya Dimitrova; Elisa Ognibene; Antonio Caprioli; Elena V. Schmalhausen; Vladimir I. Muronetz; Luciano Saso (pp. 826-832).

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the β-amyloid precursor protein (βAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with β-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon γ (INFγ). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of β-amyloid, or thiorphan, an inhibitor of neprilysin involved in β-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNγ, which can influence the progression of the AD, significantly reduced the enzyme activity.

Keywords: Glyceraldehyde-3-phosphate dehydrogenase; Catalytic activity; Alzheimer disease; Animal models

A mushroom lectin from ascomycete Cordyceps militaris by Eui Cha Jung; Ki Don Kim; Chan Hyung Bae; Ju Cheol Kim; Dae Kyong Kim; Ha Hyung Kim (pp. 833-838).

A mushroom lectin has been purified from ascomycete Cordyceps militaris, which is one of the most popular mushrooms in eastern Asia used as a nutraceutical and in traditional Chinese medicine. This lectin, designated CML, exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes. SDS-PAGE of CML revealed a single band with a molecular mass of 31.0 kDa under both nonreducing and reducing conditions that was stained by silver nitrate, and a 31.4 kDa peak in a Superdex-200 HR gel-filtration column. The hemagglutination activity was inhibited by sialoglycoproteins, but not in by mono- or disaccharides, asialoglycoproteins, or de- O-acetylated glycoprotein. The activity was maximal at pH 6.0–9.1 and at temperatures below 50 °C. Circular dichroism spectrum analysis revealed that CML comprises 27% α-helix, 12% β-sheets, 29% β-turns, and 32% random coils. Its binding specificity and secondary structure are similar to those of a fungal lectin from Arthrobotrys oligospora. However, the N-terminal amino acid sequence of CML differs greatly from those of other lectins. CML exhibits mitogenic activity against mouse splenocytes.

Keywords: Ascomycete; Mushroom; Cordyceps militaris; Lectin

Mucolytic activity of bacterial and human chitinases by Niek N. Sanders; Vincent G.H. Eijsink; Petra S. van den Pangaart; R.J. Joost van Neerven; Peter J. Simons; Stefaan C. De Smedt; Joseph Demeester (pp. 839-846).

Several pulmonary pathologies, like cystic fibrosis (CF), are characterized by hypersecretion and stasis of tenacious mucus. Bacterial glycosidases are known to degrade mucins but their use as mucolytic agents is questionable. The observation that bacterial chitinases degrade mucins and the recent discovery of human chitinases, which have been proposed to be involved in the genesis of asthma, prompted us to evaluate the mucolytic properties of human derived chitinases. The effect of these human chitinases, and bacterial chitinases (positive control), on the viscoelasticity of CF sputa and on the electrophoretic mobility of human mucins was tested. Commercial bacterial chitinase drastically degraded CF sputum, while human derived chitinases did not. Accordingly, the commercial bacterial chitinase was found to degrade mucins, whereas recombinant human chitinases did not. A thorough analysis of the commercial chitinase elucidated that contaminating proteases and also nucleases assisted in the mucolytic effect. Indeed, recombinant bacterial chitinases very slightly reduced the viscoelasticity of CF sputum, but they caused a significant degradation of the CF sputum when they were combined with proteases. In conclusion, this work shows that recombinant human and recombinant bacterial chitinases have no or very low mucolytic activities, respectively. The observed mucolytic properties of commercial bacterial chitinase are due to a synergistic effect between chitinolytic and proteolytic enzymes at one hand and at the other hand also due to the presence of contaminating nucleases.

Keywords: Abbreviations; AMCase; acidic mammalian chitinase; BSA; bovine serum albumin; ChiA; chitinase A from; Serratia marcescens; ChiB; chitinase B from; Serratia marcescens; CF; cystic fibrosis; COPD; chronic obstructive pulmonary disease; FIP; fédération internationale pharmaceutique; G′; elasticity modulus; G″; viscosity modulus; Gal; galactose; GalNAc; N; -acetylgalactosamine; GlcNAc; N; -acetylglucosamine; NANA; N; -acetylneuraminic acid; rhDNase-I; recombinant human deoxyribonuclease-I; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresis; PBS; phosphate buffered saline; 4-MU-(GlcNAc).; ₂; 4-methylumbelliferyl β-; d; -; N; ,; N; ′-diacetylchitobioseMucus; Cystic fibrosis; Mucin; Respiratory disease; Chitinase

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