BBA - General Subjects (v.1624, #1-3)

Calcium distributions in human hair by ToF-SIMS by I.M. Kempson; W.M. Skinner; P.K. Kirkbride (1-5).
Calcium distributions on internal and external surfaces of longitudinally sectioned hairs were analysed with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Externally, calcium deposits were observed at the cuticle scale edges. Internal sections showed that the bulk of calcium exists within or just inside the cuticle layer. The medulla may or may not be enriched and other localised concentrations exist in one of two forms; either associated with granular structures or the hair proteins. Calcium appears to show an affinity for proteins with low sulfur content.
Keywords: Section; Localisation; Melanin; Protein; SEM-EDX;

FT-IR study of heterologous protein expression in recombinant Escherichia coli strains by Diletta Ami; Loredana Bonecchi; Simona Calı̀; Gaetano Orsini; Giancarlo Tonon; Silvia Maria Doglia (6-10).
Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy. A marker band at 1628 cm−1 allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way. The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation.
Keywords: Inclusion body; FT-IR microspectroscopy; Escherichia coli; Expression of heterologous protein; Interferon alpha;

Diagnosis of enzyme inhibition based on the degree of inhibition by Fernando Antunes; H.Susana Marinho; M.Carmo Barreto; M.Leonor Pavão; Ruy E. Pinto (11-20).
In this work, a method for the diagnosis of kinetic inhibition, based on the dependence of the degree of inhibition (ε i) on the inhibitor concentration [I] and on the substrate concentration [S], is presented. Because the degree of inhibition is a ratio between rates, kinetic data are normalized by the introduction of an internal control—the rate of the uninhibited reaction. Therefore, the error associated with the kinetic measurements decreases and less experimental measurements are necessary to achieve the diagnosis. The process described, which uses graphical and/or non-linear fitting procedures, allows distinguishing between 20 different kinds of inhibition, including not only linear and hyperbolic, but also parabolic and rational 2,2 inhibitions. Rational 2,2 indicates a new type of inhibition corresponding to an incomplete parabolic inhibition, i.e. mechanistically it corresponds to an inhibitor that binds to two inhibition sites producing enzymatic complexes that are still active. In spite of its comprehensiveness, the diagnosis process is greatly facilitated by the division of the diagnosis of the inhibition in a step-by-step procedure, where only two rival models are evaluated in each step. In the non-linear fittings, the choice between rival models uses a test based on information statistics theory, the Akaike information criterion test, in order to penalize complex models that tend to be favoured in fittings. Finally, equations that allow the determination of inhibition kinetic constants were also deduced. The formalism presented was tested by examining inhibition of acid phosphatase by phosphate (a linear competitive inhibitor).
Keywords: Linear and hyperbolic inhibition; Parabolic and rational 2,2 inhibition; Acid phosphatase;

We explored the possibility to apply single-domain antibodies from Camelidae for immunoaffinity purification of the ice structuring protein (ISP) from Lolium perenne, which modifies ice crystal growth and therefore has potential application in medicine, biotechnology, agriculture and (frozen) foods. Using phage display together with an appropriate selection method, a group of candidate fragments was isolated from a llama-derived immune library. Affinity chromatography using a purposely selected antibody coupled to a matrix yielded a completely pure and functional ISP. Due to the extreme refolding capabilities and physical stability of single-domain antibodies, the affinity matrix could be regenerated more than 2000 times without loss of capacity, while the fragment's monomeric nature permitted an efficient elution of antigen. The results of this study show that highly pure proteins can be recovered from biological material in a single-step process.
Keywords: Single-domain antibody fragment; Phage display; Immunoaffinity chromatography; Ice structuring protein; Protein purification;

Identification of Translin/Trax complex as a glucose response element binding protein in liver by Ru Feng Wu; Kiyoshi Osatomi; Lance S. Terada; Kosaku Uyeda (29-35).
Previously, we found a novel protein factor in the livers of rats fed a high-carbohydrate diet, which binds to the major late transcription factor (MLTF)-like site within the glucose response element (GRE) of the liver-type pyruvate kinase (L-PK) gene [J. Biol. Chem. 274 (1999) 1100]. This factor, termed glucose response element binding protein (GRBP), exists in both liver cytosol and nucleus. In order to identify GRBP, we purified to homogeneity cytosolic GRBP from rat liver extract and identified it as a Translin/Trax heteromeric complex. Based on partial amino acid sequences, we have cloned full-length rat cDNAs of both Translin and Trax. The nuclear and the cytosolic Translin/Trax complex were both large polymers of 240 and 420 kDa, respectively. The molar ratio of Translin/Trax in the polymers was 2:1 in the liver cytosols. The nuclear and cytosolic Translin/Trax complexes as well as expressed His-tagged Translin bound to double- and single-stranded MLTF sites of the GRE of L-PK gene more avidly than to single-stranded Bcl-CL1, which was initially thought to be specific for Translin. Our findings indicate that the Translin/Trax complex constitutes the previously described GRBP, and that this complex binds the GRE of the L-PK gene with high affinity. The precise physiologic role of GRBP, however, remains unclear.
Keywords: Glucose response element binding protein; Liver-type pyruvate kinase; Translin; Trax; Carbohydrate;

Expression and secretion of ficolin β by porcine neutrophils by Andrew S. Brooks; Jutta Hammermueller; Josepha P. DeLay; M.Anthony Hayes (36-45).
Ficolins are collagenous lectins that bind N-acetylglucosamine (GlcNAc) as well as some bacterial surfaces, and may have opsonic and complement-activating functions. Ficolin α in porcine plasma binds Actinobacillus pleuropneumoniae serotype 5 (APP) in a GlcNAc-dependent manner. In the present study, we discovered that porcine neutrophils, but not platelets or mononuclear cells, contained a different ficolin that migrated as a 39-kDa band on SDS-PAGE and resembled a minor component of plasma ficolins that binds APP. However, neutrophil ficolins (pI range 6.4–7.4) were readily distinguished from plasma ficolin α (pI 5.2–5.8) by 2D PAGE. Neutrophil ficolin was consistent with ficolin β by pI and peptide mass fingerprinting with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Porcine neutrophils expressed ficolin β, but not ficolin α, as determined by RT-PCR. Ficolin β was present in the membrane and cytoplasmic fractions of nonactivated neutrophils, but the majority of ficolin β was secreted upon activation with PMA. Ficolin α readily bound to intact APP, but ficolin β did not under the same conditions. These studies demonstrate that neutrophils express ficolin β and secrete it when activated; however, ficolin β may have different binding functions than ficolin α.
Keywords: Ficolin; Neutrophil; Actinobacillus pleuropneumoniae; Lectin; Pig;

The pathway for the biosynthesis of cysteine and homocysteine in Methanococcus jannaschii has been examined using a gas chromatography-mass spectrometry (GC-MS) stable isotope dilution method to identify and quantitate the intermediates in the pathways. The first step in the pathway, and the one responsible for incorporation of sulfur into both cysteine and methionine, is the reaction between O-phosphohomoserine and a presently unidentified sulfur source present in cell extracts, to produce l-homocysteine. This sulfur source was shown not to be sulfide. The resulting l-homocysteine then reacts with O-phosphoserine to form l-cystathionine, which is cleaved to l-cysteine. The pathway has elements of both the plant and mammalian pathways in that the sulfur is first incorporated into homocysteine using O-phosphohomoserine as the acceptor and the resulting homocysteine, via transsulfuration, supplies the sulfur for cysteine formation. The pathway leading to these two amino acids represents an example of metabolic thrift where the preexisting cellular metabolites O-phosphohomoserine and O-phosphoserine are used as the ultimate source of the carbon framework for the biosynthesis of these amino acids. These findings explain the absence of identifiable genes in the genome of this organism for the biosynthesis of cysteine and homocysteine.
Keywords: Methanococcus jannaschii; l-cysteine; Homocysteine;

Combination effects of complement regulatory proteins and anti-complement polymer by Yasuo Yoshioka; Ryo Suzuki; Takayuki Okamoto; Naoki Okada; Yohei Mukai; Hiroko Shibata; Yasuo Tsutsumi; Natsuki Dohi; Noriko Okada; Shinsaku Nakagawa; Tadanori Mayumi (54-59).
We previously reported the development of a “cytomedicine” that consists of cells trapped in alginate–poly-l-lysine–alginate (APA) microcapsules and agarose microbeads. The functional cells that are entrapped in semipermeable polymer are completely isolated from cellular immune system. However, the ability of cytomedicine to isolate cells from the humoral immune system, which plays an essential role in xenograft rejection, is low. Therefore, the goal of the present study was to develop a novel cytomedicine that could protect the entrapped cells from injury of the complement system. We investigated the applicability of the complement regulatory protein (CRP), Crry, to cytomedicine. Crry-transfected cells entrapped within agarose microbeads resisted injury by complement to a degree, while entrapment of Crry transfected cells within agarose microbeads containing polyvinyl sulfate (PVS), a novel cytomedical device with anti-complement activity, clearly protected against complement attack. These data indicate that the combination of a CRP and a cytomedical device with anti-complement activity is a superior device for cytomedical therapy.
Keywords: Complement; Regulatory protein; Polymer;

Characterization of a trypsin-like serine protease of activated B cells mediating the cleavage of surface proteins by Anna Biró; Zoltán Hérincs; Erzsébet Fellinger; László Szilágyi; Zsuzsa Barad; János Gergely; László Gráf; Gabriella Sármay (60-69).
Activated B cells may cleave their surface receptors due to the proteolytic activity on the cell membrane or in its vicinity. We attempted to isolate and characterize the protease(s) responsible for this cleavage. Zymograms prepared from the supernatant and the plasma membrane fraction of activated human B cells and BL41/95 cell line exhibited a 85–90 kDa doublet band with protease activity, while that of resting B cells did not. Soybean trypsin inhibitor (STI), Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) and EDTA treatment abolished the activity of this protease. The excess of Zn2+ ions in EDTA did not restore the enzymatic activity, while it was completely recovered in the presence of Ca2+. We affinity-purified a 85–90 kDa protease from the supernatant of BL41/95 cells using STI coupled to Sepharose 4B beads, and measured its kinetic parameters. For the arginyl substrate K M was 358±59 μM and for the lysyl substrate 582±103 μM. TLCK and benzamidine inhibited the protease at micromolar, while STI at nanomolar concentrations. Both the inhibition profile and the substrate specificity suggest that it is a trypsin-like serine protease. We assume that the 85–90 kDa serine protease expressed on and secreted by activated B cells and BL41/95 cell line is responsible for the cleavage of various membrane proteins, including Fcγ receptors; thus it may play a crucial role in regulating B cell's function.
Keywords: Serine protease; Human; B cell; Shedding;

A cold-adapted endo-arabinanase from Penicillium chrysogenum by T. Sakamoto; H. Ihara; S. Kozaki; H. Kawasaki (70-75).
Previously, three arabinan-degrading enzymes were isolated from Penicillium chrysogenum 31B. Here we describe another arabinan-degrading enzyme, termed Abnc, from the culture filtrate of the same organism. Analysis of the reaction products of debranched arabinan by high-performance anion-exchange chromatography (HPAEC) revealed that Abnc cleaved the substrate in an endo manner and that the final major product was arabinotriose. The molecular mass of Abnc was estimated to be 35 kDa by SDS-PAGE. Enzyme activity of Abnc was highest at pH 6.0 to 7.0. The enzyme was stable up to 30 °C and showed optimum activity at 30 to 40 °C. Compared with a mesophilic counterpart from Aspergillus niger, Abnc exhibited a lower thermal stability and optimum enzyme activity at lower temperatures. Production of Abnc in P. chrysogenum was found to be strongly induced by arabinose-containing polymers and required a longer culture time than did other arabinanase isozymes in this strain.
Keywords: Cold-adapted enzyme; Endo-arabinanase; Penicillium chrysogenum;

Growth and membrane polarization of Pseudomonas aeruginosa UG2 cells grown under randomized microgravity (RMG) and 1×g were measured in a high aspect ratio vessel (HARV) and also in batch cultures mixed at 12 and 150 rpm in Erlenmeyer shake flasks. Membrane polarization was measured using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). No differences were observed in the growth curves or membrane polarization values (about 0.300) under all three culture conditions. However, the net effect of RMG at the single cell level may be still unknown. It may be possible that RMG effects are species-dependent or bacterial cells with a small mass and volume may be near the threshold where RMG exerts a minimal effect.
Keywords: Bacteria; Fluorescent probe; Growth; Membrane fluidity; Membrane polarization; Pseudomonas aeruginosa; Randomized microgravity (RMG);

In this work, the effect of Fenton reaction on two elastin cross-linked amino acids, desmosine (DES) and isodesmosine (IDE), in the absence or presence of different wavelength radiations generated from artificial sources has been evaluated using LC/ESI-MS. Irradiation as well as incubation of DES or IDE solutions in the presence of Fe2+ and H2O2 resulted in products with m/z 497.1 and 481.1 for [M+H]+. A strongly dose-dependent degradation of both amino acids was observed upon exposure to UVB at doses ranging from 0 to 3 J/cm2 and a moderate dose-dependent degradation upon exposure to UVA at doses 10 times higher than that of UVB. A significant time-dependent degradation of DES and IDE was also observed upon exposure of these amino acids to a lamp emitting visible light similar to sunlight. Exposure of both amino acids to IR radiation (520 W) for 8 h did not cause significant degradation.
Keywords: Desmosine; Isodesmosine; Skin; Elastin; LC/MS; Radiation;

Ammonia, produced by bacterial degradation of unabsorbed and endogenous nitrogenous compounds, is found to be present at millimolar concentrations in the colon lumen. From in vivo animal experiments, this metabolite has been shown to alter colonic epithelial cell morphology and to increase compensatory cell proliferation when present in excess. In this in vitro study, using the human colon adenocarcinoma HT-29 Glc−/+ cell line treated with increasing doses of NH4Cl, we found that 20 mM NH4Cl, a concentration close to that found in the large intestine lumen, was able to increase the volume of vacuolar lysosomes and to repress HT-29 Glc−/+ cell proliferation. This growth-inhibitory effect was not correlated with decrease of cell viability, with modification of cell differentiation and change of the cell distribution in the different cell cycle phases, thus indicating a proportional slowdown in all cell cycle phases. In contrast to what is found in healthy colonocytes, ammonia was not metabolized by HT-29 cells into carbamoyl-phosphate (carbamoyl-P) and citrulline, indicating that ammonia was likely acting on cells by itself. This agent was shown to markedly reduce cellular ornithine decarboxylase (ODC) activity resulting in a threefold decrease in the capacity of HT-29 cells to synthetize polyamines, these latter metabolites being strictly necessary for cell growth. The unexpected finding that ammonia is acting as an antimitotic agent against tumoral HT-29 colonic cells may be related to the inability of these cells to metabolize this compound.
Keywords: Ammonia; Colon carcinoma cell; Metabolism; Polyamine; Proliferation;

Cyclin-dependent kinase 11p110 activity in the absence of CK2 by Nancy A Sachs; Richard R Vaillancourt (98-108).
Cyclin-dependent kinase (CDK)11p110, formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. To further evaluate the regulation of CDK11p110 catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11p110 from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. These results were extended through the observation that CDK11p110 serves as a substrate for CK2 and the identification of a phosphorylation site on CDK11p110 at Ser227 by LC-MS/MS. To obtain CDK11p110 devoid of CK2, CDK11p110 was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the amino-terminus of CDK11p110. Recombinant CDK11p110 was purified from the media and phosphorylation of histone H1 subsequently demonstrated. After demonstrating retention of CDK11p110 kinase activity, it was evaluated for activity on the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II), but only CK2 was found to phosphorylate the CTD.
Keywords: CDK11p110; CK2; LC-MS/MS; CTD; Phosphorylation; High five insect cell;

Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate by Akinori Ogawa; Nobuyuki Sumitomo; Mitsuyoshi Okuda; Katsuhisa Saeki; Shuji Kawai; Tohru Kobayashi; Susumu Ito (109-114).
A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45°C. The enzyme was rapidly inactivated by incubation over 45°C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.
Keywords: Subtilisin; Serine protease; Cloning; Alkaliphilic; Bacillus;

Photosensitized inactivation of T7 phage as surrogate of non-enveloped DNA viruses: efficiency and mechanism of action by M. Egyeki; G. Turóczy; Zs. Majer; K. Tóth; A. Fekete; Ph. Maillard; G. Csı́k (115-124).
We investigated the efficiency and the mechanism of action of a tetraphenyl porphyrin derivative in its photoreaction with T7 phage as surrogate of non-enveloped DNA viruses. TPFP was able to sensitize the photoinactivation of T7 phage in spite of the lack of its binding to the nucleoprotein complex. The efficiency of TPFP photosensitization was limited by the aggregation and by the photobleaching of porphyrin molecules. Addition of sodium azide or 1,3-dimethyl-2-thiourea (DMTU) to the reaction mixture moderated T7 inactivation, however, neither of them inhibited T7 inactivation completely. This result suggests that both Type I and Type II reaction play a role in the virus inactivation. Optical melting studies revealed structural changes in the protein part but not in the DNA of the photochemically treated nucleoprotein complex. Polymerase chain reaction (PCR) also failed to demonstrate any DNA damage. Circular dichroism (CD) spectra of photosensitized nucleoprotein complex indicated changes in the secondary structure of both the DNA and proteins. We suggest that damages in the protein capsid and/or loosening of protein–DNA interaction can be responsible for the photodynamic inactivation of T7 phage. The alterations in DNA secondary structure might be the result of photochemical damage in phage capsid proteins.
Keywords: Tetraphenyl porphyrin; Photodynamic virus inactivation; T7 phage;

Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria by Yoshiro Nakajima; Jun Ishibashi; Fumiko Yukuhiro; Ai Asaoka; DeMar Taylor; Minoru Yamakawa (125-130).
Defensins are a major group of antimicrobial peptides and are found widely in vertebrates, invertebrates and plants. Invertebrate defensins have been identified from insects, scorpions, mussels and ticks. In this study, chemically synthesized tick defensin was used to further investigate the activity spectrum and mode of action of natural tick defensin. Synthetic tick defensin showed antibacterial activity against many Gram-positive bacteria but not Gram-negative bacteria and low hemolytic activity, characteristic of invertebrate defensins. Furthermore, bactericidal activity against pathogenic Gram-positive bacteria including Bacillus cereus, Enterococcus faecalis and methicillin-resistant Staphylococcus aureus was observed. However, more than 30 min was necessary for tick defensin to completely kill bacteria. The interaction of tick defensin with the bacterial cytoplasmic membrane and its ability to disrupt the membrane potential was analyzed. Tick defensin was able to disrupt the membrane potential over a period of 30–60 min consistent with its relatively slow killing. Transmission electron microscopy of Micrococcus luteus treated with tick defensin showed lysis of the cytoplasmic membrane and leakage of cellular cytoplasmic contents. These findings suggest that the primary mechanism of action of tick defensin is bacterial cytoplasmic membrane lysis. In addition, incomplete cell division with multiple cross-wall formation was occasionally seen in tick defensin-treated bacteria showing pleiotropic secondary effects of tick defensin.
Keywords: Antibacterial peptide; Cytoplasmic membrane permeabilization; Defensin; Tick;

The interaction of lithotripter-generated shock waves with adherent cells is investigated using high-speed optical techniques. We show that shock waves permeabilize adherent cells in vitro through the action of cavitation bubbles. The bubbles are formed in the trailing tensile pulse of a lithotripter-generated shock wave where the pressure drops below the vapor pressure. Upon collapse of cavitation bubbles, a strong flow field is generated which accounts for two effects: first, detachment of cells from the substrate; and second, the temporary opening of cell membranes followed by molecular uptake, a process called sonoporation. Comparison of observed cell detachment with results from a theoretical model considering peeling cell detachment by a wall jet-induced shear stress shows reasonable agreement.
Keywords: Sonoporation; Drug delivery; Cell detachment; Shock wave; Cavitation;

We have examined by immunoblotting the effect of three oxidant compounds on the level of hepatic elongation factor-2 (eEF-2). Rat liver homogenates were exposed to cumene hydroperoxide (CH), 2-2′-azobis (2-aminopropane) dihydrochloride (AAPH) and H2O2. Only CH treatment produced the disappearance of eEF-2, probably due to a phenomena of peptide bond cleavage. The direct implication of free radical species in this process is evident because of the fact that the inclusion of a free radical scavenger such as melatonin prevented the eEF-2 depletion. The results also suggest that the disappearance of eEF-2 induced by CH can be linked to a lipid peroxidant process, which could account for the decline of protein synthesis in aging and other circumstances where lipid peroxidation is high.
Keywords: Elongation factor-2; Cumene hydroperoxide; AAPH; Hydrogen peroxide; Melatonin; Lipid peroxidation;