bluuebunny/crossref_metadata_2025 huggingface dataset:
The geostationary field during dayside erosion events 1996–2001: A joint Wind, ACE, and GOES study
Authors: Mühlbachler Stefan , Farrugia Charles J. , Biernat Helfried K. , Torbert Roy B.
Published: December 2003

We carry out a statistical investigation on dayside magnetosphere erosion during the period 1996–2001, using Wind, ACE, and GOES observations supplemented by ground‐based magnetograms. The inner magnetospheric signature of erosion that we focus on is the depression of the geostationary magnetic field strength (ΔBtot) within 2 hours of local magnetic noon. We relate this decrease to the southward component of the interplanetary magnetic field (IMF) in the range −16 ≤ Bz ≲ 0 nT and the solar wind dynamic pressure in the range ∼0–6 nPa. Thus both relevant interplanetary parameters extend to well beyond average values at 1 AU. The geostationary field strength (Btot) is derived from measurements by the GOES 8, 9, and 10 spacecraft. Our data set consists of 288 measurements. We adopt two approaches. In one approach, we first correct for the effect of dynamic pressure on the geostationary field strength by studying days with various dynamic pressure values but low IMF clock angles, θ (<45°). With the latter condition we mean to exclude dayside reconnection to a reasonable degree. We then use this pressure correction to infer the erosion contribution to Btot (ΔBtot). We find ΔBtot[nT] = −3.58 + 1.67Bz with a correlation coefficient of 0.59. This analysis is most reliable in giving the change of the geostationary field to a decrease in IMF Bz, for which we obtain ΔBtot/Bz = −1.67 nT per 1 nT decrease in Bz. In the second approach, we do a bivariate fit of Btot to the (Bz, Pdyn) data pairs and obtain Btot[nT] = 97.64 + 20.22 + 1.56Bz. Major sources of error are discussed, and the results are compared to previous work.
Uptake of neutral polar vapor molecules by charged clusters/particles: Enhancement due to dipole‐charge interaction
Authors: Nadykto Alexey B. , Yu Fangqun
Published: December 2003

Recently proposed ion‐mediated nucleation theory suggests that the enhanced uptake of neutral polar molecules by the charged clusters as a result of dipole‐charge interaction may be critical to the nucleation of particles in the atmosphere. A rigorous parameterization of the enhancement effect of dipole‐charge interaction on the growth of charged clusters is presented and the important parameters controlling this enhancement factors are explored. The interaction of polar gas molecules with charges significantly enhances the growth rate of small charged clusters/particles. Polar molecules with higher dipole moment and smaller molecular size have larger enhancement factor. The enhancement factor decreases with increasing particle size and is roughly inversely proportional to temperature when the charged particles are small. At T of 300 K, the enhancement factor reaches up to 10 for the uptake of sulfuric acid molecules by ions with diameter of ∼0.5 nm but decreases quickly to 2 for uptake by a charged particle of ∼2 nm in diameter. For the uptake by molecular ions, the enhancement factor can reach as high as 20 for water molecules in the mesopause and as high as 15 for sulfuric acid in the polar stratosphere. The enhancement factors for the uptake of water dimers by charged clusters are consistently higher (up to 20–25%) than the corresponding enhancement factors for monomers. The hydration of sulfuric acid molecules in the atmosphere may affect the enhancement factor and hence ion‐mediated nucleation rate, which remains to be investigated.
Herpesviruses in human periodontal disease
Authors: Contreras Adolfo , Slots Jørgen
Published: December 2003

Recent studies have identified various herpesviruses in human periodontal disease. Epstein–Barr virus type 1 (EBV‐1) infects periodontal B‐lymphocytes and human cytomegalovirus (HCMV) infects periodontal monocytes/macrophages and T‐lymphocytes. EBV‐1, HCMV and other herpesviruses are present more frequently in periodontitis lesions and acute necrotizing ulcerative gingivitis‐lesions than in gingivitis or periodontally healthy sites. Reactivation of HCMV in periodontitis lesions tends to be associated with progressing periodontal disease. Herpesvirus‐associated periodontitis lesions harbor elevated levels of periodontopathic bacteria, including Acrinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteriodes forsythus, Prevotella intermedia, Prevotella nigrescens and Treponema denticola. It may be that active periodontal herpesvirus infection impairs periodontal defenses, thereby permitting subgingival overgrowth of periodontopathic bacteria. Alteration between latent and active herpesvirus infection in the periodontium might lead to transient local immunosuppression and explain in part the episodic progressive nature of human periodontitis. Tissue tropism of herpesvirus infections might help explain the localized pattern of tissue destruction in periodontitis. Absence of herpesvirus infection or viral reactivation might explain why some individuals carry periodontopathic bacteria while still maintaining periodontal health. Further studies are warranted to delineate whether the proposed herpesvirus‐periodontopathic bacteria model might account for some of the pathogenic features of human periodontal disease.
Effects of the anti‐androgen finasteride on 5α‐reduction of androgens in the presence of progesterone in human gingival fibroblasts: modulatory actions of the alkaline phosphatase inhibitor levamisole
Authors: Tilakaratne A. , Soory M.
Published: December 2003

Oestrogens and androgens stimulate collagen matrix synthesis, while progesterone is a competitive inhibitor for the 5α‐reduction of testosterone to 5α‐dihydrotestosterone (DHT). The anti‐androgen finasteride is a specific inhibitor of the 5α‐reductase type 2 isoenzyme, associated with anabolic functions. The aim of this investigation is to study the effects of progesterone and finasteride on 5α‐reduction of androgen substrates by human gingival fibroblasts. Monolayer cultures of human gingival fibroblasts (HGF) of the 4th–9th passage were established in Eagle's minimum essential medium (MEM). Duplicate incubations were performed with 14C‐testosterone/14C‐4‐androstenedione as substrates and progesterone (P) or finasteride (F), at concentrations of 0.5, 1, 3 and 5 μg/ml, alone and in combination, for 24 h. Similarly, the effects of the alkaline phosphatase inhibitor levamisole (L, 30 μg/ml) and P were studied. Steroid metabolites were analysed and quantified, using a radioisotope scanner. Progesterone inhibited DHT synthesis in HGF from 14C‐testosterone by 24–62%(n=8; p<0.01). Finasteride caused 59–82% inhibition (n=8; p<0.01). The combination of P+F showed a similar degree of inhibition (68–78%) of DHT synthesis to that of F alone (n=8; p<0.01). There was 35–56% stimulation of 17β‐HSD (hydroxysteroid dehydrogenase) activity by P, F and P+F (n=8; p<0.01). When 14C‐4‐androstenedione was used as substrate there was 47% inhibition of 5α‐reductase activity at higher concentrations of P and 63 and 44% stimulation at 0.5 and 1 μg/ml (n=8; p<0.01). F and P+F caused 40–67% inhibition of this activity. P, F and P+F caused 2–2.7‐fold stimulation of 17β‐HSD activity in response to all concentrations studied. L inhibited DHT synthesis from both substrates by 36–38%, with further inhibition of 55–70%(n=4; p<0.01), with P; this is suggestive of ligand‐independent alkaline phosphatase activity mediated by 5α‐reductase. Inhibition of 5α‐reductase activity by finasteride in gingival fibroblasts is suggestive of target tissue anabolic functions in gingivae and competitive inhibition by progesterone, is suggestive of regulation of hormone mediated tissue responses during repair.
Developmental changes of the vasculature in the periodontal ligament of rat molars: a scanning electron microscopic study of microcorrosion casts
Authors: Tsukada H. , Ishikawa H. , Nakamura S. , Yoshida S.
Published: December 2003

Vascularization of the periodontal ligament was examined in developing upper first molars of rats from 5 to 30 d after birth with light and scanning electron microscopy. Formation of the vascular network in the periodontal ligament (PDL) started with the beginning of root formation. The PDL vessels derived from the basal region of the tooth germ ran parallel to the long axis of the root and connected with the vascular network of the enamel organ at the cervical end. The boundary of these 2 networks was initially indistinct but became clearer with the progress of root formation. The PDL vessels further elongated longitudinally and connected with each other by lateral branches to form a coarse mesh. Other vessels derived from the alveolar bone via Volkman's canals also contributed to the vascular construction of the PDL. The vessels from the alveolar bone provided branches to the existing mesh of the PDL. Consequently, the vascular network of the PDL consisted of vessels from 2 sources: 1 derived from the basal region of the tooth germ, and the other from the alveolar bone. The density of the vascular network reduced with the progress of root formation, especially at the middle part of the root, but the mesh at the apical region maintained a basket‐like structure.
Salivary matrix metalloproteinase (MMP‐8) levels and gelatinase (MMP‐9) activities in patients with type 2 diabetes mellitus
Authors: Collin Hanna‐Leena , Sorsa Timo , Meurman Jukka H. , Niskanen Leo , Salo Tuula , Rönkä Hanne , Konttinen Yrjö T. , Koivisto Anna‐Maija , Uusitupa Matti
Published: December 2003

We studied the salivary levels and activities of the matrix metalloproteinases (MMP)‐8 and ‐9 in 45 type 2 diabetic patients and 77 control subjects. The patients' mean glycosylated haemoglobin (HbAlc) was 8.7%, indicating an unsatisfactory metabolic control of the disease. The MMP levels were further related to the clinical and microbiological periodontal findings as well as to salivary flow rate and other factors. The salivary flow rate, albumin and amylase concentrations were similar in type 2 diabetic patients to those in the control group. The mean gingival and periodontal pocket indexes were higher in the diabetes group. The number of potential periodontopathogenic bacteria was lower, however, in the diabetic than in the control group. Zymography and immunoblotting revealed that the major MMPs in the type 2 diabetic patients’ saliva were MMP‐8 and MMP‐9. Salivary MMP levels and activities in type 2 diabetic patients were in general similar to those in the control group. However, the correlation coefficients using multiple regression analysis revealed that gingival bleeding, pocket depths and HbAlc were associated with increased MMP‐8 levels which, in turn, were negatively predicted by elevated plasma lipid peroxide levels in the diabetic group. Our data on salivary MMP‐8 and ‐9 do not support the concept of generalized neutrophil dysfunction in unbalanced diabetes. Moreover, plasma lipid peroxidation levels reflecting the increased oxidative burden, which is generated mainly by triggered neutrophils, do not indicate neutrophil dysfunction due to diabetes, but may rather be related to the increased tissue damage in an uncontrolled disease. However, advanced periodontitis in type 2 diabetes seems to be related to elevated salivary MMP‐8 levels which might be useful in monitoring periodontal disease in diabetes.
The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status
Authors: Könönen Eija , Wolf Juhani , Mättö Jaana , Frandsen Ellen V. G. , Poulsen Knud , Jousimies‐Somer Hannele , Asikainen Sirkka
Published: December 2003

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first‐named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS‐PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother–child pairs were analyzed by arbitrarily primed PCR (AP‐PCR). Similar AP‐PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra‐familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP‐PCR types were most obvious within periodontally healthy mother–child pairs.
Urantide: an ultrapotent urotensin II antagonist peptide in the rat aorta
Authors: Patacchini Riccardo , Santicioli Paolo , Giuliani Sandro , Grieco Paolo , Novellino Ettore , Rovero Paolo , Maggi Carlo Alberto
Published: November 2003

In this study we describe the ability of two human urotensin‐II (hU‐II) derivatives [Pen5,Orn8]hU‐II(4–11) and [Pen5,DTrp7,Orn8]hU‐II(4–11) (urantide) to block hU‐II‐induced contractions in the rat isolated thoracic aorta. Both compounds competitively antagonized hU‐II‐ induced effects with pKB=7.4±0.06 (n=12) and pKB=8.3±0.09 (n=12), respectively. In contrast, neither [Pen5,Orn8]hU‐II(4–11) nor urantide (1 μM each) was able to modify noradrenaline‐ or endothelin 1‐induced contractile effects. At micromolar concentrations, [Pen5,Orn8]hU‐II(4–11) produced weak (25% of hU‐II maximum) agonist responses in the rat aorta, whereas urantide was totally uneffective as agonist up to 1 μM. In addition, [Pen5,Orn8]hU‐II(4–11) and urantide displaced [125I]urotensin II from specific binding at hU‐II recombinant receptors (UT receptors) transfected into CHO/K1 cells (pKi=7.7±0.05, n=4 and pKi=8.3±0.04, n=4, respectively). To our knowledge, urantide is the most potent UT receptor antagonist so far described, and might represent a useful tool for exploring the (patho)physiological role of hU‐II in the mammalian cardiovascular system.

British Journal of Pharmacology (2003) 140, 1155–1158. doi:10.1038/sj.bjp.0705555
Increased effects of C‐type natriuretic peptide on contractility and calcium regulation in murine hearts overexpressing cyclic GMP‐dependent protein kinase I
Authors: Wollert Kai C , Yurukova Sevdalina , Kilic Ana , Begrow Frank , Fiedler Beate , Gambaryan Stepan , Walter Ulrich , Lohmann Suzanne M , Kuhn Michaela
Published: November 2003

C‐type natriuretic peptide (CNP) and its receptor guanylyl cyclase (GC‐B) are expressed in the heart and modulate cardiac contractility in a cGMP‐dependent manner. Since the distal cellular signalling pathways remain unclear, we evaluated the peptide effects on cardiac function and calcium regulation in wild‐type (WT) and transgenic mice with cardiac overexpression of cGMP‐dependent protein kinase I (PKG ITG).

In isolated, perfused working WT hearts, CNP (10 nM) provoked an immediate increase in the maximal rates of contraction and relaxation, a small increase in the left ventricular systolic pressure and a decrease in the time of relaxation. These changes in cardiac function were accompanied by a marked increase in the levels of Ser16‐phosphorylated phospholamban (PLB).

In PKG ITG hearts, the effects of CNP on cardiac contractility and relaxation as well as on PLB phosphorylation were markedly enhanced.

CNP increased cell shortening and systolic Cai2+ levels, and accelerated Cai2+ decay in isolated, Indo‐1/AM‐loaded WT cardiomyocytes, and these effects were enhanced in PKG I‐overexpressing cardiomyocytes.

8‐pCPT‐cGMP, a membrane‐permeable PKG activator, mimicked the contractile and molecular actions of CNP, the effects again being more pronounced in PKG ITG hearts. In contrast, the cardiac reponses to β‐adrenergic stimulation were not different between genotypes.

Taken together, our data indicate that PKG I is a downstream target activated by the CNP/GC‐B/cGMP‐signalling pathway in cardiac myocytes. cGMP/PKG I‐stimulated phosphorylation of PLB and subsequent activation of the sarcoplasmic reticulum Ca2+ pump appear to mediate the positive inotropic and lusitropic responses to CNP.

British Journal of Pharmacology (2003) 140, 1227–1236. doi:10.1038/sj.bjp.0705567
Expression of biologically active human butyrylcholinesterase in the cabbage looper (Trichoplusia ni)
Authors: Platteborze Peter L. , Broomfield Clarence A.
Published: November 2003

This investigation examined the utility of three recombinant protein‐expression systems (COS cells, insect cells and insect larvae) to cost‐effectively produce biologically active human butyrylcholinesterase (BuChE). It was determined that baculovirus‐infected insect cells (Sf9 and High 5) expressed 3.5‐ and 8.2‐fold, respectively, more active enzyme than COS‐7 cells. Baculovirus‐infected cabbage looper (Trichoplusia ni) insect larvae produced over 26 times more than High 5 cells; in fact, one baculovirus‐infected insect larva provided more active protein than 100 ml of insect cell culture. Analysis of the larvally expressed proteins revealed that the vast majority of BuChE expressed was inactive due to extensive degradation that occurred in vivo. However, the active form of BuChE does have enzyme kinetics similar to those of its human serum counterpart. Cabbage looper larvae were also examined for their ability to serve as an in vivo animal model to study protection against anti‐cholinesterase toxicity. This was unsuccessful due to their high tolerance to the very toxic organophosphorus compounds tested. This tolerance may be attributed at least in part to a novel endogenous organophosphorus acid anhydride hydrolase activity that is capable of hydrolysing the chemical‐warfare nerve agents sarin (isopropyl methylphosphonofluoridate) and soman (pinacolyl methylphosphonofluoridate). These results show that cabbage looper larvae can serve as an inexpensive recombinant protein‐expression system for human BuChE.
Phase‐specific optimization of multiple endotoxin‐protein production with genetically engineered Bacillus thuringiensis
Authors: Yang Xiao‐Ming , Wang Shaw S.
Published: November 2003

An optimization approach was designed to specifically study the toxin‐expression phase of the fermentation process of a genetically engineered Bacillus thuringiensis strain expressing dual toxin proteins (CryI and CryIII). The study has resulted in the discovery of important nutrient and process factors affecting toxin‐protein yield. The results show that the existence of nitrogen sources in the medium during the toxin‐expression phase is detrimental to the toxin‐protein expression, while a high carbon‐source level (40 g/l) encouraged protein expression. The study also suggests that the depletion of nitrogen source is the trigger for B. thuringiensis to initiate sporulation and toxin expression. A temperature setting of 28 °C for B. thuringiensis fermentation processes is optimal for protein yield, and reduces the oxygen requirement. It was found that the optimal conditions for spore yield and for toxin‐protein yield were not the same, even though sporulation and toxin formation proceed simultaneously during the fermentation process. Scale‐up studies were also conducted to confirm the optimal conditions obtained from a small‐scale optimization study.
Albumin purification from human placenta
Authors: Cabrera‐Crespo Joaquin , Gonçalves Viviane Maimoni , Martins Elizabeth A. L. , Grellet Sheyla , Lopes Alexandre Paulo Yague , Raw Isaías
Published: November 2003

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin‐purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross‐flow ultrafiltration (CFUF) membrane; (iv) thermo‐coagulation at 70 °C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion‐exchange chromatography Q‐Sepharose; (viii) hydrophobic‐interaction chromatography with phenyl‐Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A403 of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.
Purification and characterization of a highly stable tyrosinase from Thermomicrobium roseum
Authors: Kong Kwang‐Hoon , Hong Min‐Pyo , Choi Sang‐Sook , Kim Yong‐Tae , Cho Sung‐Hye
Published: November 2003

Tyrosinase, with an isoelectric point at pH 4.9, was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. Gel filtration, N‐terminal amino acid sequencing and SDS/PAGE analysis indicate that T. roseum tyrosinase is composed of two identical subunits, each with a molecular mass of 43000 Da. The enzyme exhibited high substrate specificity towards catechol, chlorogenic acid, L‐3‐(3,4‐dihydroxyphenyl)‐L‐alanine (L‐DOPA) and pyrogallol. The Km value of the enzyme for L‐DOPA was 0.18 mM. β‐Mercaptoethanol and sodium diethyldithiocarbamate notably inhibited the enzymic activity. The activity of the enzyme was optimal at pH 9.5 and 70 °C, and was increased by addition of 1 mM Mg2+, K+ or Cu2+. The enzyme was highly stable against high temperature and guanidine hydrochloride. The N‐terminal amino acid sequence of the enzyme was determined to be Asp‐Ile‐Asn‐Gly‐Gly‐Gly‐Ala‐Thr‐Leu‐Pro‐Gln‐Lys‐Leu‐Tyr. These facts indicate that T. roseum tyrosinase appears to be distinct from the tyrosinases so far purified from other sources.
Introduction of thiol‐reactive structures on to soluble and insoluble proteins
Authors: Manta Carmen , Ovsejevi Karen , Betancor Lorenz , Grazú Valeria , Batistoni Julio , Batista‐Viera Francisco , Carlsson Jan
Published: November 2003

When proteins containing disulphide groups were oxidized with magnesium monoperoxyphthalate at acidic pH, they acquired the property of binding thiol compounds. This was the case with the insoluble protein keratin, chosen for having a large number of disulphide bridges, and with soluble ones like BSA and immunoglobulins. The potential applications of some of these modified proteins for the preparation of soluble bioconjugates have been explored. As a particular example of an application, the immobilization of activated IgG on to solid phases might provide a new way for preparing immunoadsorbents.
Reverse transcription of an RNA genome from databasing paper (FTA®)
Authors: Rogers Craig D. G. , Burgoyne Leigh A.
Published: November 2003

The instability of RNA in solutions during storage and travel is an impediment to its utilization in routine diagnostics. A robust and simple approach to the problem of RNA protection and processing is offered by storage of RNA desiccated with processing procedures that do not solublize the RNA until the beginning of reverse transcription. The feasibility of this general approach was tested with coxsackievirus B4 (CVB‐4) from blood or culture fluid held on a storage and transport medium (FTA®) and analysed by reverse transcriptase PCR (RT‐PCR) without removing the RNA from the FTA® until reverse transcription. Phase‐trapping techniques based on water‐miscible solvents such as ethanol or phenol were compared with simple buffers and concentrated lithium chloride solutions. RT‐PCR detection of viral RNA reached a sensitivity of ≈0.1 fg, which is comparable with other non‐nested PCR techniques. Whole blood as a virus vehicle significantly interfered with CVB‐4 detection, but to an acceptable degree. Desiccation‐storage of the RNA of CVB‐4 appears to be unaffected by weeks on the storage medium under ambient conditions. These characteristics indicate that this approach forms a credible developmental base for RNA‐based pathogen diagnostics with particular application to the problem of transporting potentially infectious body fluids to a centralized laboratory for analysis.
A bioelectrode for penicillin detection based on gluten‐membrane‐entrapped microbial cells
Authors: Chao Hsin‐Peng , Lee Wen‐Chien
Published: November 2003

A bioelectrode for penicillin detection was established using gluten membrane in which cells of Escherichia coli harbouring the plasmid pUC18 coding for penicillinase synthesis were entrapped. For the entrapment preparation, lyophilized cells were mixed with gluten solution during the formation of gel, and the resultant gel was hardened by the addition of oxidized starch. The immobilization procedure employed an inexpensive base material and avoided the use of purified enzyme. When the membrane with a thickness of 30 μm and a cell content of 33% (w/w) was used, the steady response of the bioelectrode to increasing concentrations of penicillin G buffered with 0.025 M phosphate was linear over the range of 1–16 mM. The response time was less than 3 min. Decreasing the cell content decreased the steady response due to a decline in enzymic activity. The treatment of lyophilized cells with ultrasonication or chemically with either N‐cetyl‐N,N,N‐trimethylammonium bromide or guanidine hydrochloride rendered cells permeabilized and exposed the periplasmic enzyme to penicillin. The response time became shorter, but a serious decline in steady responses was observed because of the loss of enzyme activity during the cell permeabilization. The microbial electrode also showed different responses to penicillin G solution, depending on the pH and buffer concentration.
Improvement of receptor‐mediated gene delivery to HepG2 cells using an amphiphilic gelling agent
Authors: Cho Cheong‐Weon , Cho Young‐Sik , Lee Hye‐Kyung , Il Yeom Young , Park Sue‐Nie , Yoon Do‐Young
Published: November 2003

Gene transfer was performed using asialo‐oroso mucoid–polylysine (ASOR–PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel‐electrophoretic analysis, the ASOR–PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR–PL conjugate/pCMVβ‐gal plasmid). The gene‐transfer efficiency of the ASOR–PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5‐bromo‐4‐chloroindol‐3‐yl β‐D‐galactopyranoside (‘X‐Gal’) staining and Chlorophenol Red β‐D‐galactopyranoside. When an expression vector for the tumour‐suppressor gene p53, pCMVp53, complexed to ASOR–PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza‐virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of β‐galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor‐mediated gene‐delivery system for hepatic gene therapy using ASOR–PL conjugate in combination with poloxamer 407 may be developed in the future.
A kinetic analysis using fractals of cellular analyte‐receptor binding and dissociation
Authors: Sadana Ajit , Vo‐Dinh Tuan
Published: November 2003

A fractal analysis is presented for cellular analyte‐receptor binding and dissociation kinetics using a biosensor. Data taken from the literature may be modelled, in the case of binding, using a single‐fractal analysis or a dual‐fractal analysis. The dual‐fractal analysis represents a change in the binding mechanism as the reaction progresses on the surface. The predictive relationship developed for the equilibrium constant, K (affinity which is equal to k d/k 1or2), as a function of the analyte concentration is of particular value since it provides a means by which the affinity may be manipulated. This should be of assistance in cell‐surface reactions, drug‐candidate optimization and for the design of immunodiagnostic devices. Relationships are also presented for the binding and dissociation rate coefficients as a function of their corresponding fractal dimension, D f or the degree of heterogeneity that exists on the surface, and the analyte concentration in solution. When analyte‐receptor binding or dissociation is involved, an increase in the heterogeneity on the surface (increase in D f or D fd as the case may be) leads to an increase in the binding and the dissociation rate coefficients. It is suggested that an increase in the degree of heterogeneity on the surface leads to an increase in the turbulence on the surface owing to the irregularities on the surface. This turbulence promotes mixing, minimizes diffusional limitations and leads subsequently to an increase in the binding and the dissociation rate coefficients. The binding and dissociation rate coefficients are rather sensitive to the degree of heterogeneity, D f and D fd, respectively, that exists on the biosensor surface. The heterogeneity on the surface in general affects the binding and dissociation rate coefficients differently. In general, the analyte concentration in solution has a mild affect on the fractal dimension for binding or the fractal dimension for dissociation. This is indicated by the low values of the exponent in the predictive relationships developed.
Characterization of recombinant invertase expressed in methylotrophic yeasts
Authors: Acosta Niuris , Beldarraín Alejandro , Rodríguez Luis , Alonso Yamirka
Published: November 2003

We studied, for the first time, characterization of the invertase expressed in the methylotrophic yeasts Hansenula polymorpha and Pichia pastoris in terms of enzyme conformational stability and structural behaviour induced by temperature as a function of pH using enzymic assays, differential scanning calorimetry, fluorescence and CD. The enzyme produced in both hosts was very stable over a broad range of pH values, keeping its enzymic activity and structure above 60 °C. Thermal denaturation, as measured by differential scanning calorimetry, was always irreversible. However, the fact that scanning rate had no effect on the calorimetric curves gave us the chance to analyse the data from a thermodynamic point of view. The conformational stabilities were essentially identical under the experimental conditions studied, but stability was always slightly higher in the enzyme expressed in H. polymorpha . This fact indicates that the greater degree of glycosylation of this enzyme form contributed to its increased global stability. Reactivation upon heating at 80 °C depends on protein concentration, suggesting that irreversibility could be associated with slow refolding kinetics at high protein concentration.
Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris : evidence of hyper O‐glycosylation
Authors: Letourneur Odile , Gervasi Gaspard , Gaïa Sébastien , Pagès Joëlle , Watelet Bénédicte , Jolivet Michel
Published: November 2003

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris . The truncated protein lacked the C‐terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N‐glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C‐terminal of the construction to aid purification by immobilized metal‐chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O‐linked oligosaccharides containing α1‐2‐, α1‐3‐ or α1‐6‐linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human‐serum‐derived antibodies, specific for the native SAG1, which suggested that the O‐glycosylations had no major effect on the protein conformation. However, ELISA and Western‐blot analysis with human sera showed that the O‐carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.
Using an experimental design for the optimization of LVV‐haemorphin‐7 and VV‐haemorphin‐7 extraction by an organic solvent mixture in the course of bovine haemoglobin peptic hydrolysis
Authors: Froidevaux Renato , Nedjar‐Arroume Naïma , Choisnard Luc , Bigan Muriel , Guillochon Didier
Published: November 2003

Mixture design was used to improve the extraction of two opioid peptides (LVV‐haemorphin‐7 and VV‐haemorphin‐7) by water‐immiscible solvents in the course of bovine haemoglobin peptic hydrolysis. Because of the loss of the peptic activity, these two haemorphins did not appear in the aqueous phase when the peptic reaction was achieved in the presence of butan‐2‐ol alone. We have shown that it is possible to use octan‐1‐ol, as a co‐solvent, to recover the peptic activity. However, when the hydrolysis was achieved with octan‐1‐ol alone, the two haemorphins appeared in the aqueous phase, but were not extracted by the organic phase. We therefore investigated haemorphin extraction in the course of the hydrolysis reaction by a mixture of butan‐2‐ol and octan‐1‐ol. We have determined the optimal conditions with respect to the extraction of opioid peptides and the stability of the pepsin. To design a future continuous‐stirred‐tank reactor, we propose a biphasic system composed of 45% water, 45% butan‐2‐ol and 10% octan‐1‐ol for the extraction of the two haemorphins in the course of bovine haemoglobin peptic hydrolysis.
Solution stability studies of the subunit components of meningococcal C oligosaccharide–CRM197 conjugate vaccines
Authors: Ho Mei M. , Lemercinier Xavier , Bolgiano Barbara , Crane Dennis , Corbel Michael J.
Published: November 2003

Spectroscopic methods were used to detect modifications in the structures of CRM197, the mutant diphtheria toxin, and meningococcal C capsular oligosaccharide following their conjugation and incubation at various temperatures. Meningococcal C oligosaccharide–CRM197 conjugate vaccines obtained from two different manufacturers were incubated at −20, 4, 23, 37 or 55 °C for 5 weeks or subjected to ten cycles of freeze–thawing. The CRM197 carrier protein and the saccharide components of the treated vaccines were monitored by CD and NMR spectroscopic techniques. CD data indicated incubation temperature‐dependent conformational changes in the carrier protein from vaccine A. Modifications appeared in both secondary and tertiary structures of the conjugated CRM197 when incubated at 23 °C or above. This was characteristic of the ‘open’ conformation previously observed for this protein component. The NMR spectra also indicated modification of the structure of the conjugated CRM197 component of vaccine A when incubated at 23 °C or above, but failed to show any modification in the conjugated oligosaccharide. On the other hand, the structure of the oligosaccharide chains in vaccine B appeared to be degraded following incubation at 55 °C, even though the thermal effect on the conjugated CRM197 was less apparent. Repeated freeze–thawing did not affect the CD or NMR spectra. In conclusion, the two meningococcal C oligosaccharide–CRM197 conjugate vaccines were stable when stored at their recommended temperatures, but were differently affected by elevated temperatures. The conjugates differ in their conjugation chemistry, attachment positions, oligosaccharide chain length and loading, as well as recommended pH and storage buffer, and their different stability properties can probably be attributed to a combination of these factors.
Studies on lipase‐affinity adsorption using response‐surface analysis
Authors: Kamimura Eliana Setsuko , Medieta Oscar , Rodrigues Maria Isabel , Maugeri Francisco
Published: November 2003

Lipases are widely distributed enzymes that can be obtained from animals, plants and micro‐organisms. Coupling lipases with a wide range of substrates allows the opportunity for synthesis of optically pure pharmaceutical preparations, flavour compounds and other food additives. Affinity chromatography owes its power as a purification method to specific biological interactions. Response‐surface analysis was chosen to study column efficiency. This method allows the understanding of interactions between variables with advantages over conventional methods, which involve changing one variable while fixing others at certain levels. The aim of this work was to study the influence of the ratio bed height/column diameter (L/D) and superficial velocity (V) on the column efficiency. The experimental design involved the two variables, L/D (2–10) and v (1–2 cm/min), at five levels. Lipase was obtained from Geotrichum sp. culture in a complex medium composed of 5% corn‐steep liquor, 0.5% NH4NO3 and 1% olive oil at 30 °C, with 1VVM (air volume/medium volume per min) aeration and 400 rev./min agitation. Maximum lipase activity was 19 units/ml after almost 9 h of fermentation. This lipase could potentially be used in esterification reactions to increase the content of γ‐linolenic acid and to produce bioaromas for food industries. The adsorption assays were carried out in a fixed‐bed column with an affinity adsorbent, which was obtained by reaction of a gel with oleic acid as ligand. Breakthrough curves were obtained for all experiments. It has been shown that the lower the values of both L/D and v, the higher the column efficiency (maximum 65.43%). Also, it was observed from the response surface that the efficiency reached a minimum at an L/D of around 8.
Regulated secretion of proinsulin/insulin from human hepatoma cells transduced by recombinant adeno‐associated virus
Authors: Yang Ya‐Wun , Hsieh Yuan‐Chiao
Published: November 2003

To employ hepatocytes as surrogate β‐cells for gene therapy of diabetes, a regulatory system was devised in this study by placing the human insulin cDNA under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter, followed by the cytomegalovirus immediate early promoter‐driven enhanced‐green‐fluorescent‐protein open reading frame. The expression cassette was inserted into the adeno‐associated virus vector between two inverted terminal repeats, and used to produce recombinant adeno‐associated virus (rAAV). HepG2 human hepatoma cells were transduced by rAAV at the desired multiplicity of infection, followed by treatment with various concentrations of retinoic acid, dexamethasone, dibutyryl cAMP (dbcAMP) and 3‐isobutyl‐1‐methylxanthine (IBMX). The cell‐culture media were collected at 8, 16 and 24 h later. Proinsulin/insulin levels were determined with human proinsulin/insulin radioimmunoassay kits. Transduction of HepG2 cells by rAAV showed that green fluorescence was produced as early as 12 h after rAAV infection. Flow‐cytometrical analysis demonstrated that transduction efficiency increased with the numbers of transducing rAAV particles used. The transduced hepatocytes were shown to secrete immunoreactive proinsulin/insulin, which were stimulated by the concentrations of retinoic acid, dexamethasone and dbcAMP in the culture medium. High conversion from proinsulin into insulin occurred when these cells were treated with dexamethasone and dbcAMP. The presence of IBMX enhanced the secretion of proinsulin/insulin from the dbcAMP‐treated cells. We conclude that rAAV is a promising vector for gene therapy of diabetes. Regulated secretion of proinsulin/insulin can be obtained in the rAAV‐transduced HepG2 cells conferred with the PEPCK promoter via rAAV‐mediated gene transfer.
Cold lability of lactate dehydrogenase isoenzymes and the effective preparation of reference material for clinical laboratory use
Authors: Yoshikuni Keiko , Matsuda Takashi , Xia Wu Li , Inagaki Michiko , Nishimura Mamiko , Tanishima Kiyoh
Published: November 2003

To evaluate the storage of samples and enzyme reference materials, and to improve the commutability for inter‐laboratory surveillance of activity values of lactate dehydrogenase (LD; EC 1.1.1.27) in clinical laboratory medicine and in animal veterinary medicine, we studied the electrophoretic patterns and cold lability of LD isoenzymes from tissue sources of some common vertebrate species and also from human serum sources. Among many isoenzymes from these sources, only rat LD fractions showed similar electrophoretic patterns to those of human sera, and rat LD‐1 fraction was relatively cold‐stable. Total LD and isoenzyme LD‐1 activities in routine laboratory samples and quality‐control sera were measured using eight kinds of commercially available LD assay kit, including lactate and pyruvate substrate systems. Coefficients of variation between these assay kits were markedly reduced when the activities were calculated using the partially purified rat LD‐1 fraction as an enzyme reference material, compared with the activities calculated using the factor indicated in each assay kit. In the regression analysis, the intercept and slope were calculated for the regression equations obtained from 12 pairs of these assay kits. The values obtained from a small amount of human serum and control serum samples were within the 95% confidence regions of those from larger amounts of human serum samples by using the present rat LD‐1 standard for measuring LD‐1 activities with lactate substrate. It was evident that a cold‐stable and homogeneous LD isoenzyme as an enzyme reference material might contribute to accurate measurement of activities in heterogeneous samples for inter‐laboratory quality‐assurance surveys in both human and animal clinical laboratory use.
Running‐buffer composition influences DNA–protein and protein–protein complexes detected by electrophoretic mobility‐shift assay (EMSA)
Authors: Roder Karim , Schweizer Michael
Published: November 2003

The gel‐shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid–protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non‐denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non‐specific competitor or gel concentration and/or composition. We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp1 (stimulatory protein 1), NF‐Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of glycine concentration on Sp1 binding using recombinant glutathione S‐transferase–Sp1 fusion protein expressed in Escherichia coli.
Recombinant Opc protein from Neisseria meningitidis reconstituted into liposomes elicits opsonic antibodies following immunization
Authors: Carmenate Tania , Mesa Circe , Menéndez Tamara , Falcón Viviana , Musacchio Alexis
Published: November 2003

The reconstitution of recombinant bacterial outer membrane proteins (OMPs) into their native conformations after purification has been the major problem in their use as effective vaccines. Liposomes have been shown to be an attractive approach, providing a native‐like environment for these antigens. The meningococcal recombinant Opc (rOpc) protein, produced as inclusion bodies in Escherichia coli, was incorporated into phospholipid vesicles consisting of dipalmitoyl phosphatidylcholine and cholesterol. The incorporation of rOpc into the lipid bilayer was demonstrated, and the reconstitution of some native epitopes was tested using a set of monoclonal antibodies. Subcutaneous immunization of Balb/c mice with rOpc‐containing vesicles resulted in the generation of a high level of specific antibodies. The elicited antibodies reacted with the native meningococcal protein and showed opsonic activity.
Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application
Authors: Pecher Gabriele , Schirrmann Thomas , Kaiser Lothar , Schenk Jörg A.
Published: November 2003

Dendritic cells (DCs) are the most potent antigen‐presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol®. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA‐DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.