Protein and Peptide Letters - Volume 13, Issue 2, 2006

The etiology of multiple sclerosis (MS) remains unclear. To determine if autoantibodies to myelin basic protein (MBP) are produced during parvovirus B19 infection, A competitive ELISA was performed using plasma from MS patients exhibiting high IgG titers for parvovirus. Our results showed the addition of MBP decreased the binding of IgG to B19 antigen in A dose dependent fashion suggesting A possible link between parvovirus B19 and A subset of patients with clinical MS.

Thermal unfolding kinetics of β-glucosidase B from Paenibacillus polymyxa and its thermoresistant mutant H62R were determined from far-UV circular dichroism (CD) measurements at different temperatures. The unfolding of both enzymes followed simple two-state kinetics. The new ionic pair formed between Arg62 and Glu429 in the H62R variant did not change substantially the enzyme structure as judged by far-UV CD and fluorescence spectra, but produced an increase in the unfolding activation barrier of 0.95 ± 0.10 kcal mol-1, in good agreement with the energetic contribution reported for surface salt bridges in proteins. Eyring's analysis of the unfolding kinetic constants showed that the activation enthalpies for thermal denaturation of both enzymes were essentially the same. Thus, the greater kinetic stability rendered by the salt bridge seems to be due to A reduction in the activation entropy.

We studied the processing of amyloid β-peptides (Aβs) including Aβ1-40, Aβ1-42 and pAβ3-42 by rat neutral cysteine protease bleomycin hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His14-Gln15 and Phe19-Phe20 bonds, in Aβ1-40 and Aβ1-42 by endopeptidase activity. The resultant peptides were degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Aβs were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His14-Gln15 bond in pβA3-42 within A short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Aβ1-40 and Aβ1-42 were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Aβs.

A simple nonradioactive assay, which was based on the specific shift of the absorbance maximum of methylene blue induced by its intercalation into poly(A) molecules, was developed for poly(A)-specific ribonuclease (PARN). A good linear relationship was found between the absorbance at 662 nm and the poly(A) concentration. The assay conditions, including the concentration of methylene blue, the incubation temperature and time, and the poly(A) concentration were evaluated and optimized.

(DIPP-L-Leu)2-L-LysOCH3 is A diisopropylphosphoryl dipeptide which is known to induce apoptosis of human leukemia K562 cells. The molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that (DIPP-L-Leu)2-L-LysOCH3-induced apoptosis is associated with cytosolic accumulation of cytochrome c, sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS) and elevation of intracellular Ca2+ concentration. A specific caspase assay reveals an increase in caspase-9 and caspase-3 activity but no change in caspase-8 activity. Immunofluorescence analysis indicates that (DIPP-L-Leu)2-L-LysOCH3 induced upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-2 and Bcl-xL. These results suggest that the mitochondria-regulated death pathway mediates (DIPP-L-Leu)2-L-LysOCH3-induced K562 cells apoptosis.

A novel chemically-modified insulin, ε-NB29-lipoyl insulin, was selectively prepared by the covalent linkage of α-lipoic acid (LA) to the ε-amino group of LysB29 of insulin without any protecting agent and analyzed by PAGE, HPLC, MALDI-TOF-MS. Monolipoyl- insulin maintained the glucose-lowering effect as well as native insulin and showed A longer duration of action than native insulin and an inhibitory effect towards trypsin degradation.

In this study, we calculated the amino-acid distribution rank of 1201 hemagglutinins from influenza A viruses dated from 1918 to 2004 in order to compare them with respect to subtypes, species and years. After noticing fluctuations in distribution rank along the time course, we used the fast Fourier transform to determine the mutation periodicity of the hemagglutinins. Then we estimated our position at the current cycle of hemagglutinin evolutionary process to determine how many years remain before the next possible outbreak of influenza and bird flu. Finally, we used the trend channel to outlook the future of hemagglutinins for the next half A century. As our study covers almost all the full-length amino-acid sequences of hemagglutinins from various influenza A viruses, the conclusions will be valid for years until the number of hemagglutinins in Protein Databank is significantly increased.

The recombinant chimeric enzyme, AnsB-TTP-CETPC, comprising asparaginase, tetanus toxin helper T cell epitope and human CETP B cell epitope was expressed as A soluble protein in Escherichia coli. The purified chimeric enzyme exhibited approximate 83% activity of the native asparaginase. After immunization with three doses of chimeric enzyme, high titers of anti-CETP antibodies were induced and lasted more than eighteen weeks in mice, and could even be detected at A dilution of 1:12800 by normal ELISA assay. The specificity of anti-CETP antibody was verified by Western blot assay. After displaying on the surface of asparaginase, the weak antigenicity of CETP epitope was effectively overcome, there after A strong CETP-specific immune response was evoked in mice immunized with the chimeric enzyme. Histochemical analysis of mice kidney tissue showed that immunization with the chimeric enzyme did not cause any pathological changes in mice. Collectively, the chimeric enzyme may be further developed as A vaccine against atherosclerosis in the future.

Human β-defensin-2 (hBD2) is A small cationic peptide with A broad range of antimicrobial activity. An E. coli cell-free system was employed to express the hBD2 fusion protein by using the hBD2 gene with 14 rare codons. The results showed that the expression level of trxA-hBD2 fusion protein was 0.35 mg/ml, which is the same as that obtained with A synthetic codon-optimized gene. By using another fusion partner (GFP), similar high-level expression was also achieved in this cell-free system. This meant that human beta-defensin-2 gene could be directly used to express hBD2 fusion protein efficiently in an E. coli cell-free system without the optimization of codons. The expression level of hBD2 fused with thioredoxin could be further improved up to 2.0 mg/ml by adopting A continuous exchange cell-free system. A simple one-stage affinity purification procedure was also developed to recover this fusion protein efficiently.

Quinolinate phosphoribosyltransferase was examined for susceptibility to different chemical modification reagents. Loss of enzyme activity with trinitrobenzenesulfonate (TNBS) occurred when 1.1 lysines per subunit were modified. Tryptic digestion of the modified enzyme followed by HPLC-MS analysis of the peptides showed Lys70 reacts with TNBS. Based on x-ray studies, this amino acid participates in A conformational change distant from the active site.

Using radio-receptor analysis, it has been demonstrated that human ε-casomorphin-7 (Tyr-Pro-Phe-Val- Glu-Pro-Ile) displaces 3H-spiperone from 5-HT2-receptors of rat brain frontal cortex. IC50 of human β-casomorphin-7 was 8 μM. These data suggest that one of the mechanisms of neurotropic action of β-casomorphin-7 is might be associated with its influence on the serotoninergic system.

We report A new prohormone convertase gene of insect origin, Trichoplusia ni furin, which was cloned from BTI-Tn-5B-4 (Tn5) insect cells. We constructed A truncated mutant that lacked Cys-rich repeated segments. Using baculovirus expression system and standard enzymatic assay, we obtained recombinant Tn furin and evaluated aspects of its function.

The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had A higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55°C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.

Electric field stimulation (EFS) causes excitatory non adrenergic-non cholinergic (eNANC) and cholinergic constrictions in the guinea pig isolated bronchus, the activation of eNANC and cholinergic nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2 ([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2 ([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin (NC) inhibited constrictions in isolated bronchus of guinea pig. The results show that NC (1 μmol/L) inhibited EFS-induced eNANC and cholinergic constrictions compared with the control, in which nociceptin was not applied. After pretreatment with [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2 and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, and NST did not affect the inhibitions induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2 and NST did not cause any appreciable effects on EFS-induced eNANC and cholinergic constrictions in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2 and [F/G]NC(1- 13)NH2 but not NST act as selective antagonists of the NC receptor and the effects of NC on EFS-induced constrictions of guinea pig isolated bronchus.

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6- dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on A solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4- sulfophenylsulfonyl)ethoxycarbonylamino acids.

There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.

Production of GST-fused WW domains of FBP proteins was increased using the bubbling cultivation method for E. coli. Purified WW domains of FBP11 and FBP28 bound A PL motif peptide with dissociation constants (KD) of 248 ± 27 and 1880 ± 280 μM, respectively.

Human spindlin1 is A newly screened and identified gene product related to ovarian carcinomas and is highly homologous to mouse spindlin. It is an abundant maternal transcript expressed in the mouse during the transition from oocyte to embryo. Here, the recombinant human spindlin1 has been overexpressed in Escherichia coli BL21, purified and crystallized using the hanging-drop vapour-diffusion method. Crystals diffracting to 2.25 Å resolution were obtained using ammonium sulfate as precipitant. The crystals belong to the space group P212121, with unit-cell parameters A =40.7 Å, b =84.4 Å, c =136.4 Å, α=β=γ=90° . Assuming two molecules per asymmetric unit, the solvent content is calculated to be 42.4%.

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C2221 with unit-cell parameters A = 53.2 Å, b = 104.6 Å, c = 520.0 Å, α = β = γ = 90° , MDH-NADH crystals to space group C2 with unit-cell parameters A = 51.5 Å, b = 51.5 Å, c = 256 Å, α = β = γ = 90 , and MDH-NADPH crystals to space group C2221 with unit-cell parameters A = 72, Å b = 72 Å, c = 520 Å, α = β = γ = 90° . The crystal of native MDH diffracted to 2.1 Å resolution.