J Lipid Res, 1999 May, 40(5), 881 - 92 Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane; Gargiulo CE et al.; Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism . FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import . In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake . To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs . We demonstrate for the first time that FACS is an integral membrane protein . Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane . Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP . Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells. J Biol Chem, 1999 May 7, 274(19), 13147 - 54 The reaction of trimethylamine dehydrogenase with trimethylamine; Jang MH et al.; The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process . As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases . The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C . The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10 . 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively . The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0 . 2 . The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased . The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site . The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme. J Biol Chem, 1999 May 7, 274(19), 13127 - 32 Characterization and cloning of celR, a transcriptional regulator of cellulase genes from Thermomonospora fusca; Spiridonov NA et al.; CelR, a protein that regulates transcription of cellulase genes in Thermomonospora fusca (Actinomycetaceae) was purified to homogeneity . A 6-kilobase NotI-SacI fragment of T . fusca DNA containing the celR gene was cloned into Esherichia coli and sequenced . The celR gene encodes a 340-residue polypeptide that is highly homologous to members of the GalR-LacI family of bacterial transcriptional regulators . CelR specifically binds to a 14-base pair inverted repeat, which has sequence similarity to the binding sites of other family members . This site is present in regions upstream of all six cellulase genes in T . fusca . The binding of CelR to the celE promoter is inhibited specifically by low concentrations of cellobiose (0.2-0.5 mM), the major end product of cellulases . The other sugars tested did not affect binding at equivalent or 50-fold higher concentrations . The results suggest that CelR may act as a repressor, and that the mechanism of induction involves a direct interaction of CelR with cellobiose. Ned Tijdschr Geneeskd, 1999 Feb 6, 143(6), 306 - 8 {Children hospitalized with acute gastroenteritis . II . No relation between clinical symptoms and causative agents isolated from feces}; Tjon A Ten WE; OBJECTIVE: To determine if it is possible to predict a bacterial cause in children with gastroenteritis on the basis of clinical features and if the number of stool cultures can be reduced . DESIGN: Retrospective . METHOD: The results of the stool cultures of 227 children admitted with acute gastroenteritis to the department of Paediatrics of Sint Joseph Hospital, Veldhoven, the Netherlands, in the period 1992-1996 were related by review of medical records to clinical symptoms and blood tests . The diagnostic values of the various parameters were calculated . RESULTS: Rotavirus was identified in 40% of the faeces of the children, other viruses in 10% and bacteria in 8% . In 95% the bacterial pathogen was identified in the first stool sample . The diagnostic value of the investigated parameters was low . CONCLUSION: It is not possible on the basis of clinical parameters to predict a bacterial gastroenteritis . The number of stool samples may, however, be reduced if the stools of each child with acute diarrhoea are first investigated for rotavirus . Only if this test is negative are further investigations required . The number of bacterial cultures can be limited to one stool sample. Anaesthesiol Reanim, 1999, 24(1), 4 - 12 {Acute failure of the intestinal barrier--pathophysiology, diagnosis, prophylaxis and therapy}; Hachenberg T et al.; The gut not only serves as a main target for the detrimental effects of stress during and after surgery, but may also promote the development of multiple organ failure after different types of severe shock . According to a current hypothesis, an impaired intestinal barrier function is associated with a decreased separation of intraluminal bacteria and toxins and systemic circulation, which may induce sepsis and multiple organ failure . Hypoperfusion during shock, reperfusion injury of the splanchnic mucosa, alterations of the micro-ecology of the gut and immunologic and hormonal disturbances are important underlying pathophysiological mechanisms . Various therapeutic concepts have been proposed such as improvement of splanchnic perfusion, nutritive and metabolic treatment by means of immunomodulating nutrients, parenteral substitution of glutamine, early onset of enteral nutrition, normalization of gut motility and selective decontamination of the gut . However, no clinical study to date could clearly demonstrate a key role of the gut in the pathogenesis of sepsis and multiple organ failure . Likewise, the efficacy of different prophylactic and therapeutic procedures remain to be studied . An aggressive treatment of shock and avoidance of microcirculatory disturbances are of principal importance for prophylaxis of multiple organ failure. J Prosthet Dent, 1999 May, 81(5), 597 - 609 Mechanical properties of dental luting cements; Li ZC et al.; STATEMENT OF PROBLEM: Dental luting cements fail by microcrack formation and bacterial ingress or by gross failure and crown dislodgment . Both of these failure modes are related to mechanical properties and deformation . PURPOSE: This study evaluated those mechanical properties of cements . METHODS AND MATERIAL . Elastic modulus for 8 representative cements (zinc phosphate, polycarboxylate, glass ionomer, encapsulated glass ionomer, resin-modified glass ionomer, resin composite, and adhesive resin composite) was measured by using a nondestructive technique and evaluated for cement type and storage time (1 hour, 1 day, 1 week, 1 month, 1 year) by 2-way ANOVA (P <.05) . Compressive properties (proportional limit, resilience, and toughness), ultimate strengths (compressive, diametral tensile, and flexural), and flexural toughness were determined and evaluated by 2-way ANOVA for 2 crosshead testing rates (5 and 0.5 mm/min) and cement type (P <.05) . RESULTS: Cements varied with respect to elastic moduli, compressive proportional limit, compressive resilience, compressive strength, compressive toughness, diametral tensile strength, flexural strength, and flexural toughness . Storage time affected the elastic moduli of different materials in different ways . Elastic moduli of polycarboxylate and glass ionomer cements increased over time, whereas the other materials changed little after the first day . Crosshead rate only significantly affected compressive proportional limit and resilience . CONCLUSIONS: Luting cements differed considerably with respect to mechanical properties. Biochemistry, 1999 Apr 27, 38(17), 5651 - 8 Proteasome activator 11S REG or PA28: recombinant REG alpha/REG beta hetero-oligomers are heptamers; Zhang Z et al.; The proteasome activator 11S REG or PA28 is a conical molecule composed of two homologous subunits, REG alpha and REG beta . Recombinant REG alpha forms a heptamer, whereas recombinant REG beta is a monomer . When mixed with REG beta, a monomeric REG alpha mutant (N50Y) forms an active hetero-oligomer in which the molar ratio of REG beta to REG alpha(N50Y) is close to 1.3 . This apparent stoichiometry is consistent with the REG alpha(N50Y)/REG beta hetero-oligomer being a heptamer composed of three alpha and four beta subunits . Chemical cross-linking of the alpha/beta oligomers revealed the presence of REG alpha-REG beta and REG beta-REG beta dimers, but REG alpha-REG alpha dimers were not detected . The mass of the REG alpha(N50Y)/REG beta hetero-oligomer determined by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) is 194 871 +/- 40 Da in good agreement with the theoretical mass of 194 856 Da for an alpha 3 beta 4 heptamer . Hexamers were not observed in the mass spectrum . For wild-type REG subunits coexpressed in bacteria cells at an apparent beta/alpha molar ratio of approximately 1.2, the resulting hetero-oligomers observed by ESI-TOF MS were again predominantly alpha 3 beta 4 heptamers, with trace amounts of alpha 4 beta heptamers also present . On the other hand, the mass spectrum contained a mixture of alpha 7, alpha 6 beta 1, alpha 5 beta 2, and alpha 4 beta 3 heptamers when the REG beta/REG alpha ratio was 0.1 . Thus, formation of heptamers is an intrinsic property of recombinant REG alpha and REG beta subunits . On the basis of these results, we propose that 11S REG purified directly from eukaryotic cells is also heptameric, likely alpha 3 beta 4 or a mixture of alpha 3 beta 4 and alpha 4 beta 3 species. Biochemistry, 1999 Apr 27, 38(17), 5596 - 602 Pleckstrin homology domain 1 of mouse alpha 1-syntrophin binds phosphatidylinositol 4,5-bisphosphate; Chockalingam PS et al.; Mouse alpha 1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to bind phosphatidylinositol 4, 5-bisphosphate (PtdIns4,5P2) . Half-maximal binding occurred at 1.9 microM PtdIns4,5P2 and when 1.2 PtdIns4,5P2 were added per syntrophin . Binding was specific for PtdIns4,5P2 and did not occur with six other tested lipids including the similar phosphatidylinositol 4-phosphate . Binding was localized to the N-terminal pleckstrin homology domain (PH1); the second, C-terminal PH2 domain did not bind lipids . Key residues in PtdIns4,5P2 binding to a PH domain were found to be conserved in alpha-syntrophins' PH1 domains and absent in PH2 domains, suggesting a molecular basis for binding. Microbiology, 1999 Apr, 145 ( Pt 4), 935 - 47 Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine; Milcamps A et al.; Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine . The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine . Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases . Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival . This is believed to be the first report of a hmgA-homologous gene in bacteria. J Chromatogr B Biomed Sci Appl, 1999 Mar 19, 724(2), 265 - 74 Formation and identification of azaarene transformation products from aquatic invertebrate and algal metabolism; de Voogt P et al.; The metabolism of two azaarenes, viz . acridine and phenanthridine, by aquatic organisms was studied in short-term and chronic laboratory tests . The identity of metabolites observed in the test waters was investigated with different analytical methods, including HPLC, GC and hyphenated LC- or GC-MS . The Zebra mussel (Dreissena polymorpha), one green alga species (Selenastrum capricornutum) and periphyton or bacteria transformed acridine into 9{10H}-acridinone . Phenanthridine was transformed into 5{6H}-phenanthridinone by midge (Chironomus riparius) larvae . The findings indicate that closely related isomers may undergo species-specific biotransformation . It was concluded that keto-metabolites are major products in the aquatic fate of benzoquinolines, which may be overlooked in the risk assessment of parent compounds . This study illustrates the typical problems with, as well as the potency of, chromatographic methods in the elucidation of metabolic routes of organic contaminants. Oral Microbiol Immunol, 1999 Apr, 14(2), 117 - 21 Restriction fragment-length polymorphism analysis of 16S ribosomal RNA genes amplified by polymerase chain reaction for rapid identification of cultivable oral treponemes; Sato T et al.; Although oral treponemes are among the most frequently found bacteria in periodontal pockets, identification of these organisms can be difficult . In this study, restriction fragment-length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA genes (16S rRNA gene PCR-RFLP) was used to generate restriction profiles of reference strains of oral treponemes including Treponema denticola, Treponema socranskii, Treponema vincentii . Treponema pectinovorum and Treponema medium as well as for Treponema phagedenis and Treponema pailidum and five treponeme strains isolated from human peridontal pockets . Before RFLP analysis, the 16S rRNA gene sequences were obtained from the GenBank database, and the analysis of the theoretical banding patterns for HpaII suggested good species discrimination . 16S rRNA gene sequences were amplified from isolated genomic DNA samples by PCR with spirochete-specific primers . The PCR products were then purified and characterized by single digestion with restriction endonuclease HpaII, and this allowed discrimination between the respective reference strains . Five clinical isolates, four T . denticola and one T . socranskii, were assigned on the basic of their restriction profiles by digestion with HpaII . 16S rRNA gene PCR-RFLP using HpaII is a rapid and reliable method for differentiation of cultivable oral treponemes. Oral Microbiol Immunol, 1999 Apr, 14(2), 109 - 16 Binding of the periodontal pathogen Actinobacillus actinomycetemcomitans to extracellular matrix proteins; Mintz KP et al.; The interaction of Actinobacillus actinomycetemcomitans, an important pathogen implicated in juvenile and adult periodontitis, with collagenous and noncollagenous proteins of the extracellular matrix was investigated . A . actinomycetemcomitans SUNY 465 bound to immobilized type I, II, III and V but not type IV collagen . Binding to immobilized collagen was saturable and concentration dependent . This interaction could not be inhibited by soluble collagen, suggesting that binding was dependent on a specific collagen conformation . Bacteria grown anaerobically exhibited decreased collagen-binding activity as compared with organisms grown acrobically . Bacterial outer membrane proteins were essential for binding to collagen . A actinomycetemcomitans SUNY 465 also bound to immobilized fibronectin . In contrast, bacteria did not bind to fibrinogen, bone sialoprotein, alpha 2-HS glycoprotein or albumin . The mechanism of the interaction with fibronectin was more complex, possibly involving both protein and nonproteinaceous components . The majority of other A . actinomycetemcomitans strains tested bound to extracellular matrix proteins in a manner similar to SUNY 465 but with minor variation . These results demonstrate that A . actinomycetemcomitans binds to proteins found in connective tissue . The interaction with extracellular matrix proteins may contribute to the virulence of this pathogen at oral and extraoral sites of infection. Nucleic Acids Res, 1999 May 15, 27(10), 2063 - 71 Comparative sequence analysis of tmRNA; Zwieb C et al.; Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences . The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence . Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions. Ophthalmic Surg Lasers, 1999 Apr, 30(4), 295 - 8 A study of the incidence of culture-positive endophthalmitis after cataract surgery in an ambulatory care center; Bohigian GM; BACKGROUND AND OBJECTIVE: To determine the incidence of culture-positive endophthalmitis after cataract surgery in an ambulatory surgical care center and to analyze the effectiveness of povidoneiodine solution in the preoperative preparation in preventing culture-positive endophthalmitis . PATIENTS AND METHODS: A retrospective series of 19,269 consecutive cases of cataract extraction with lens implantation over 12 years in an ambulatory care center was reviewed . RESULTS: Nine cases of culture-positive endophthalmitis occurred, for an incidence of 0.05% . The initial 4,740 cases (1985-1989) were performed without the use of povidone-iodine; the following 14,529 cases (1990-1996) were done using povidone-iodine . The incidence of culture-positive endophthalmitis was 0.08% and 0.03%, respectively . CONCLUSION: The incidence of culture-positive endophthalmitis in this series was very low . The use of 5% povidone-iodine, topically, appeared to be beneficial in reducing the incidence of culture-positive endophthalmitis after cataract extraction (P=0.24), but was not statistically significant in this retrospective series . Evaluation and methods to prevent endophthalmitis are difficult in retrospective clinical studies due to multiple variables and the rarity of this complication. Monaldi Arch Chest Dis, 1999 Feb, 54(1), 22 - 37 Mechanisms of ventilation-induced lung injury: physiological rationale to prevent it; Verbrugge SJ et al.; It is being increasingly realized that modes of mechanical ventilation that result in end-inspiratory alveolar overstretching and/or repeated alveolar collapse and re-expansion disturb the normal fluid balance across the alveolocapillary membrane . The effects of this include disturbance of the integrity of the endothelium and epithelium and impairment of the surfactant system and are similar to those seen in acute respiratory distress syndrome (ARDS) . There is now also evidence that these modes of mechanical ventilation may result in the translocation of bacteria from the lungs into the bloodstream and the release of inflammatory mediators from the lung tissue into the systemic circulation . It may thus be speculated that mechanical ventilation may contribute to the development of multiple organ failure (MOF) . Therefore, during mechanical ventilation, alveolar overstretching and the repeated collapse and re-expansion of alveoli should be prevented by ventilation modes that open up the lung and keep the lung open and ventilate with the smallest possible pressure amplitude . For the future, monitoring techniques should be developed that can evaluate, on-line, whether or not these therapeutic directives are being achieved. Acta Neurol Belg, 1999 Mar, 99(1), 57 - 60 Nitric oxide synthase expression in neurogenic bladder disease: a pilot study; De Ridder D et al.; Neurogenic bladders are susceptible to bladder cancer development, especially in the case of chronic indwelling catheters . The classic carcinogenesis theory involves the formation of carcinogenic nitrosamines by bacteria due to chronic infection . We designed a pilot study to evaluate the expression of the of Nitric Oxide Synthase (NOS) isoforms in bladder tissue to study the role of the endogenous formation of NO (Nitric Oxide) in neurogenic bladders . Immunohistochemistry was performed on bladder biopsies from neurogenic, normal, and obstructed bladders . The neurogenic bladder had a higher expression of endothelial NOS (eNOS), but especially of neuronal NOS (nNOS) . Besides, this extra-neuronal expression of nNOS by urothelium and interstitial cells was observed . This study proves the important role of endogenous formation of NO by suburothelial nerves but also by urothelium and interstitial cells . This overexpression could possibly be a factor in the higher incidence of bladder cancer in neurogenic bladders . On the other hand, it shows the plasticity of the NO pathways in these cases and raises important research questions concerning the physiological role of these changes. J Bacteriol, 1999 May, 181(9), 2797 - 801 Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase; Shah VK et al.; The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1 . In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH) . The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component . Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components . Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co . In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX . Complementation of these assays with extracts of A . vinelandii DJ42 . 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity . These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis . The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor. Biochim Biophys Acta, 1999 Apr 21, 1411(1), 206 - 13 Characterization of the photoreduction of the secondary quinone QB in the photosynthetic reaction center from rhodobacter capsulatus with FTIR spectroscopy Nabedryk E. The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter (Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H2O and 2H2O . The Q-B/QB FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone-protein interactions . As expected from the conservation of most of the amino acids near QB in the RCs from Rb . capsulatus and Rb . sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm-1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb . sphaeroides RCs . However, noticeable changes are observed at 1709 cm-1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm-1 (side chain and/or backbone), around 1540 cm-1 (amide II), and in the semiquinone absorption range . This FTIR study demonstrates that the environment of the secondary quinone in Rb . capsulatus is close but not identical to that in Rb . sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near QB. Vaccine, 1999 Apr 9, 17(15-16), 1846 - 57 Immunogenicity of peptides derived from a fibronectin-binding protein of S . aureus expressed on two different plant viruses; Brennan FR et al.; The D2 peptide derived from an S . aureus fibronectin-binding protein (FnBP) was expressed on the surface of the icosahedral cowpea mosaic virus (amino acids 1-30 of D2) or on the rod-shaped potato virus X (amino acids 1-38 of D2), termed CPMV-MAST1 and PVX-MAST8, respectively . Mice and rats were immunized subcutaneously with CPMV-MAST1 and mice with PVX-MAST8 in adjuvant and high titres of FnBP-specific antibody were obtained . The mouse IgG was predominantly of the IgG2a and IgG2b isotypes, which strongly bound complement component C1q, suggesting a TH1-bias in the peptide-specific responses . Sera from mice and rats immunized with CPMV-MAST1 and from mice immunized with PVX-MAST8 were shown to completely inhibit the binding of fibronectin to immobilised recombinant FnBP and rat sera against CPMV-MAST1 were able to block adherence of S . aureus to fibronectin . These studies demonstrate that the D2 peptide is highly immunogenic when expressed on 2 different plant viruses and highlight the potential of plant virus-based vaccines to protect against S . aureus infections. Microbiology, 1999 Mar, 145 ( Pt 3), 743 - 53 Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp . strain RF-1; Huang TC et al.; Sixteen nif and 'nif-associated' genes (expressed only under conditions of nitrogen fixation) in Synechococcus sp . strain RF-1 have been cloned and sequenced . All of the nif and nif-associated genes identified in Synechococcus RF-1 were arranged in a continuous cluster spanning approximately 18 kb and containing seven operons . The nifH operon (nifH-nifD-nifK) has been reported previously . nifB, fdxN, nifS, nifU and nifP were found to be located upstream of the nifH operon . nifB-fdxN-nifS-nifU were expressed as an operon . A nifP-like gene was found to be located just upstream of nifB . nifE, nifN, nifX, nifW and the nif-associated hesA, hesB and 'fdx' were found to be located downstream from nifK . The genes located downstream from nifK are arranged nifE-nifN-nifX-orf-nifW-hesA-hesB-'+ ++fdx' and span approximately 7 kb . The function of the ORF situated between nifX and nifW is not known . However, it was identified as a counterpart of ORF-2 in Anabaena sp . strain PCC 7120 based on the deduced amino acid sequence . Northern hybridization and primer extension analysis indicated that the nif and nif-associated genes are organized in nifE-nifN, nifX-orf, nifW-hesA-hesB and 'fdx'-containing operons, respectively . According to the results of this study and previous reports, the genes are expressed in a rhythmic pattern with peaks during the dark phase when the culture is grown in a 12 h light/12 h dark regimen . The rhythm persisted after the culture was transferred to continuous illumination. Int J Biochem Cell Biol, 1999 Jan, 31(1), 139 - 49 Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50; Evdokimova VM et al.; p50, the major core protein of messenger ribonucleoprotein particles (mRNPs), is a universal protein found exclusively in association with different mRNA species in the cytoplasm of somatic mammalian cells . Furthermore, p50 is the most abundant and tightly bound protein within both inactive mRNPs and active mRNPs derived from polysomes, although the latter contain a lower level of p50 . Recent experiments have revealed that, depending on the p50 to mRNA ratio, p50 may either act as a repressor or an activator of protein synthesis . On the other hand, p50 exhibits about 98% amino acid sequence identity to mammalian transcription factors that bind specifically to Y-box containing DNA . Thus, it is a counterpart of the Y-box binding proteins which are found in bacteria, plants and animals, exhibiting multiple biological activities ranging from transcriptional regulation of a wide variety of genes to 'masking' mRNA activity in germinal cells . This review summarizes our current knowledge of p50 structure and function . It also discusses the biological roles of p50 and related proteins in gene expression and describes the likely mechanisms of their action. Avian Dis, 1999 Jan-Mar, 43(1), 98 - 105 Detection of avian adenovirus by polymerase chain reaction; Xie Z et al.; An avian adenovirus-specific polymerase chain reaction was developed . The origin of primers was from the DNA sequence data of the chicken embryo lethal orphan avian adenovirus virus genome . An avian adenovirus-specific 421-bp DNA product was amplified by these primers from group I of adenovirus containing 12 serotypes and serotypes of adenovirus from group II and group III . The adenovirus-specific DNA product was also amplified from the 19 field isolates of avian adenoviruses but not from the mammalian adenovirus and other avian pathogenic viruses and bacteria . As little as 1 fg of avian adenovirus DNA was detected by gel electrophoresis and Southern blot analysis. Avian Dis, 1999 Jan-Mar, 43(1), 1 - 7 Investigations on different Ornithobacterium rhinotracheale "ORT" isolates; Hafez HM et al.; The aim of the present investigation was to determine the antigenic relationship between different Ornithobacterium rhinotracheale (ORT) isolates and to serotype field isolates obtained from turkey and chickens . Different antigen extractions (heat-stable, proteinase K-stable {lipopolysaccharide}, and sodium dodecyl sulfate {SDS} extractions) were prepared from each serotype (A, B, C, D, E, and G) as well as from 21 ORT field isolates and examined in agar gel precipitation (AGP) and enzyme-linked immunosorbent assay (ELISA) tests . The field isolates were cultured from turkey (16 isolates) and chicken (5 isolates) flocks showing respiratory manifestations . Monospecific reactions were obtained with heat-stable as well as proteinase K-stable antigens prepared from serotypes A, C, D, E, and G in AGP tests . On the other hand, with the same antigen preparations from a strain of serotype B in AGP tests, cross-reactions with antisera prepared against serotypes A and E could be detected . The cross-reactions were observed mostly between 48 and 72 hr . In applications of SDS-antigen preparations in AGP tests, cross-reactions between all serotypes except serotype C were detected between 24 and 72 hr . Testing all antigen preparation in ELISA, different cross-reactions were observed and the evaluation of the results is very difficult . Serotyping of the field isolates in AGP tests by using heat-extracted antigens showed after 24 hr that 10 out of 16 isolates from turkey belonged to serotype B, five to serotype A, and one to serotype E . Results obtained after 48-72 hr revealed cross-reactions between serotype B and E in 11 cases and between A and B in two cases . All five isolates obtained from chicken reacted after 24 hr only with serum against serotype A . After 48-72 hr, two isolates showed cross-reaction with antiserum against serotype B . Similar results were obtained with proteinase K-stable antigen. Gene, 1999 Apr 16, 230(2), 187 - 95 Characterization of a maize heat-shock protein 101 gene, HSP101, encoding a ClpB/Hsp100 protein homologue; Nieto-Sotelo J et al.; Heat shock protein 101 (HSP101) cDNA and genomic clones from maize have been isolated . The structure of maize HSP101 reveals the presence of six exons interrupted by five introns . Maize HSP101 contains a predicted open reading frame that translates into a 912-aa sequence with a mass of 101kDa . Initiation of transcription was mapped 146 bases upstream of the AUG codon . Five heat shock element (HSE) boxes were found within the proximal 289 bases of the promoter region . Southern blot analysis of genomic DNA indicates that the maize genome contains only one copy of HSP101 . A protein sequence comparison showed that maize Hsp101 belongs to the heat shock 100kDa and caseino-lytic protease B protein family (Hsp100/ClpB) that plays important roles in bacteria and yeast in the survival to extremely high temperatures and the control of proteolysis . Accumulation of HSP101 mRNA was strong under heat shock conditions, but not detectable after cold or osmotic stress treatments or by exogenous application of ABA . The analysis of the predicted supersecondary structure of maize Hsp101 showed that a coiled-coil located in the middle region of the protein is evolutionarily conserved in all members of the Clp A, B and C subfamilies . It is proposed that these supersecondary structures may have important roles in Clp function. Mutat Res, 1999 Apr 6, 425(2), 213 - 24 Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver; Enzmann H et al.; Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria . The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA) . Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA . We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo . The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo{4.3.0}non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al . {K.D . Bremm, U . Petersen, K.G . Metzger, R . Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387} was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg . The embryos were removed from the eggs after 4 days and liver samples were shock frozen . Mitochondrial DNA was purified from samples of the embryonic liver . The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA . Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear) . The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA {H . Enzmann, C . Kuhlem, E . Loser, P . Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res . 329 (1995) 113-120} . After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect . NDEA (25 mg) was used as positive control . Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA . Biochim Biophys Acta, 1999 Apr 21, 1411(1), 134 - 41 The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex; Gomes CM et al.; The succinate dehydrogenase complex of the thermoacidophilic archaeon Acidianus ambivalens was investigated kinetically and by EPR spectroscopy in its most intact form, i.e., membrane bound . Here it is shown that this respiratory complex has an unusual iron-sulfur cluster composition in respect to that of the canonical succinate dehydrogenases known . The spectroscopic studies show that center S3, the succinate responsive {3Fe-4S}1+/0 cluster of succinate dehydrogenases, is not present in membranes prepared from aerobically grown A . ambivalens, nor in partially purified complex fractions . On the other hand, EPR features associated to the remaining centers, clusters S1 ({2Fe-2S}1+/2+) and S2 ({4Fe-4S}2+/1+), could be observed . Similar findings were made in other archaea, namely Acidianus infernus and Sulfolobus solfataricus . Kinetic investigations showed that the A . ambivalens enzyme is reversible, capable of operating as a fumarate reductase - a required activity if this obligate autotroph performs CO2 fixation via a reductive citric acid cycle . Sequencing of the sdh operon confirmed the spectroscopic data . Center S3 ({3Fe-4S}) is indeed replaced by a second {4Fe-4S} center, by incorporation of an additional cysteine, at the cysteine cluster binding motif (CxxYxxCxxxC-->CxxCxxCxxxC) . Genomic analysis shows that genes encoding for succinate dehydrogenases similar to the ones here outlined are also present in bacteria, which may indicate a novel family of succinate/fumarate oxidoreductases, spread among the Archaea and Bacteria domains. Blood, 1999 May 1, 93(9), 3140 - 7 Prevention of transfusion-associated graft-versus-host disease by photochemical treatment; Grass JA et al.; Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates . The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model . Recipient mice in four treatment groups were transfused with 10(8) splenic leukocytes . (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 micromol/L S-59 and 2.1 J/cm2 UVA . Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin . Mice in the control group remained healthy and free of detectable disease . Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia . In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 micromol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD . Using an in vitro T-cell proliferation assay, greater than 10(5.1) murine T cells were inactivated by PCT . Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD. J Vasc Res, 1999 Mar-Apr, 36(2), 83 - 90 The emergence of physiological genomics; Cowley AW Jr; 'Physiological genomics' represents a research paradigm shift emerging to define the functions of tens of thousands of newly discovered genes which are expected to emerge from the sequencing of the human genome and other model organisms . Genomic tools, which will allow a higher efficiency of identification of gene function, are being developed at remarkable speed . This article discusses some of the genomic and bioinformatic tools currently available or under development to provide the infrastructure for mapping and identification of gene function in simple organisms (bacteria, zebrafish, fly, worm) and complex mammalian organisms (mouse and rat) . The problems facing the scientific community in the implementation of this functional approach are discussed as it is now evident that new technological and organizational infrastructures are emerging to link genes to overall function of whole organisms. Eur J Biochem, 1999 Apr, 261(2), 475 - 80 A methenyl tetrahydromethanopterin cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in Methylobacterium extorquens AM1; Pomper BK et al.; Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units . In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1) . Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence . The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa . Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea . The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa . Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases . Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M . extorquens AM1. Eur J Biochem, 1999 Apr, 261(2), 459 - 67 NMR studies of subunit c of the ATP synthase from Propionigenium modestum in dodecylsulphate micelles; Matthey U et al.; The structure of the Na+, Li+ or H+-binding c subunit of the ATP synthase from Propionigenium modestum was studied by NMR . Subunit c in dodecylsulphate micelles consists of four alpha-helical segments, I-IV, that are connected by short linker peptides with non-regular secondary structures . We propose that helices I (V4-I26) and IV (I69-V85) are membrane-spanning structures, and that helices II and III and the intervening hydrophilic loop are located in the cytoplasm . The Na+-binding residues Q32, E65 and S66 are located in the I-->II and III-->IV helix connections, probably near the membrane surface on the cytoplasmic side. FEBS Lett, 1999 Mar 26, 447(2-3), 131 - 4 A second mechanism of respiratory control; Kadenbach B et al.; According to the chemosmotic hypothesis, ATP is synthesized in mitochondria, bacteria and chloroplasts via the proton motive force delta p, the energy-rich intermediate of electron transport and photosynthetic phosphorylation . The general applicability of the chemosmotic hypothesis, however, was disputed until present . In particular the relationship between the rate of respiration and delta p in mitochondria was found variable, depending on the experimental conditions . Recently, a new mechanism of respiratory control was found, based on binding of ATP or ADP to subunit IV of cytochrome c oxidase, which is independent of delta p and could explain many previous results contradicting the chemosmotic hypothesis. Biochimie, 1999 Jan-Feb, 81(1-2), 139 - 46 Effect of DNA lesions on transcription elongation; Tornaletti S et al.; Some types of damage to cellular DNA have been shown to interfere with the essential transactions of replication and transcription . Not only may the translocation of the polymerase be arrested at the site of the lesion but the bound protein may encumber recognition of the lesion by repair enzymes . In the case of transcription a subpathway of excision repair, termed transcription-coupled repair (TCR) has been shown to operate on lesions in the transcribed strands of expressed genes in bacteria, yeast, mammalian cells and a number of other organisms . Certain genes in mammalian cells (e.g., CSA and CSB) have been uniquely implicated in TCR while others (e.g., XPC-HR23 and XPE) have been shown to operate in the global genomic pathway of nucleotide excision repair, but not in TCR . In order to understand the mechanism of TCR it is important to learn how an RNA polymerase elongation complex interacts with a damaged DNA template . That relationship is explored for different lesions and different RNA polymerase systems in this article. Bull Soc Pathol Exot, 1999 Feb, 92(1), 6 - 8 {Prevalence of anti-Chlamydia pneumoniae antibodies in preadolescent children in Congo}; Kabeya BK et al.; Chlamydia pneumoniae (CPn) is recognised as a common cause of both upper and lower respiratory tract infections . Seroepidemiological studies seem to indicate a world-wide distribution of this organism . In order to evaluate the prevalence of antibodies to CPn in a healthy pediatric population, we measured anti-C . pneumoniae antibodies in a group of 253 infants without respiratory tract infections, aged from 1 to 12 years . Sera were obtained from children seen at immunization clinics and schools in Lubumbashi (Congo) . Antibodies to CPn were evaluated using micro-immunofluorescence assay . IgG antibody to CPn in a titre 16 was considered as positive . The antibody prevalence was found to be 25.7% . This prevalence was 6.2% in children aged from 1 to 6 years, and 37.8% in children aged from 7 to 12 years . It was less than 10% under five years, increasing to 50% at 12 years . The progressive increasing of seropositivity related to age suggests that reinfections may be frequent . This study shows an important spread of this bacteria in the preadolescent population of an African country. Nihon Kokyuki Gakkai Zasshi, 1999 Feb, 37(2), 125 - 9 {Pulmonary nocardiosis associated with Cushing's syndrome}; Dohchin A et al.; A 54-year-old man was admitted for further investigation of multiple nodules disclosed by a chest roentgenogram . Adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome was diagnosed because serum ACTH and serum cortisol levels were elevated with a loss of diurnal rhythm . Because several extensive examinations, including inferior petrosal sinus sampling, did not detest ACTH-producing tumors, the patient was also given a diagnosis of occult ectopic ACTH syndrome . The nodules disclosed on chest roentgenograms increased gradually in size and number, and some were cavitary . Bronchial secretion samples obtained by fiberoptic bronchoscopy contained numerous Nocardia asteroides bacteria . After treatment with sulfamethoxazole-trimethoprim, the nodules gradually disappeared, leaving only scars . Although mitotane had been continuously administered to inhibit the synthesis of intrinsic corticosteroids, pulmonary nocardiosis relapsed in the patient following the termination of sulfamethoxazole-trimethoprim therapy. Kansenshogaku Zasshi, 1999 Feb, 73(2), 172 - 8 {Evaluation of mycobacteria growth indicator tube (MGIT), an automated culture system for detection of mycobacteria from clinical specimens}; Kobayashi I et al.; We compared the Mycobacteria Growth Indicator Tube 960 (MGIT 960) and Ogawa medium (OM) for the detection of mycobacteria (acid fast bacteria: AFB) using 882 sputum specimens . Overall, 120 strains of AFB were isolated by the MGIT 960 system and 99 strains of AFB were isolated by using OM . As far as Mycobacterium tuberculosis is concerned, 88 and 71 isolates were achieved by the MGIT 960 and OM respectively . A total of 28 isolates (18 isolates of M . tuberculosis and 10 isolates of nontuberculous mycobacteria: NTM) were detected by the MGIT 960 only whereas only 2 isolates (1 M . tuberculosis and 1 NTM) were detected by OM only . Of these sputum specimens, 72 were smear positive for AFB . The rates of smear negative but culture positive specimens were 8.0% (65 out of 809) for the MGIT 960 system and 6.2% (50 out of 809) for OM . The contamination rate for MGIT 960 was only 1.2% . The average time required for detection of M . tuberculosis was 14.1 days by the MGIT 960 system and 24.6 days by OM . For the NTM, the average detection time were 8.3 days for the MGIT 960 system and 22.8 days for OM . These results indicate that the MGIT 960 system allows detection of mycobacteria significantly faster than OM. Cytometry, 1999 Apr 1, 35(4), 346 - 52 Cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM) in the study of neutral lipid dynamics in Paramecium primaurelia mating types during cell line development; Ramoino P et al.; BACKGROUND: In Paramecium primaurelia, an exconjugant cell can produce two lines with different mating capacities . Mating type II cells can form a higher food vacuole number and digest the nutrient taken up in a shorter time; thus, mating type II cells grow at a faster rate than do mating type I cells . The present study was done to determine whether cells that ingest more nutrients also have a larger amount of storage lipids . METHODS: Quantitative and qualitative determinations of neutral lipids were obtained by means of cytofluorometry and fluorescence confocal laser scanning microscopy (CLSM), respectively, by using nile red on cells in different physiologic states . RESULTS: Lipid droplet number and neutral lipid content were higher in mating type II cells than in mating type I cells in the early logarithmic growth phase (i.e., immature well-fed cells) . These values were reversed during the middle and the late logarithmic phases and became equal in the stationary phase (i.e., mature starved cells) . In well-fed cells maintained with food excess, differences in neutral lipid content between the two mating types also were present in mature cells . CONCLUSIONS: Although differences between mating type I and mating type II lines were not correlated to cell size, a relation was found between lipid content and food ingestion capacity . A depletion of bacteria in the culture medium could be responsible for the lack of differences in mature starved cells . CLSM allowed us to gather volume information about the lipid droplet distribution within the cell. J Basic Clin Physiol Pharmacol, 1998, 9(2-4), 139 - 51 Novel aspects of the regulation of glycogen storage; Roach PJ et al.; The storage polysaccharide glycogen is widely distributed in nature, from bacteria to mammals . Study of its regulated accumulation has resulted in the discovery or elaboration of several important biochemical principles . Many aspects of the control of glycogen storage still remain poorly understood and glycogen metabolism continues to provide interesting models of more general relevance. Histol Histopathol, 1999 Apr, 14(2), 517 - 24 Cytological and functional aspects of telomere maintenance; Dandjinou AT et al.; The fact that eukaryotic chromosomes are linear poses a special problem for their maintenance: the natural ends of chromosomes must be distinguished from ends generated by chromosomal breakage and somehow, the chromosome ends must also be fully replicated to maintain their integrity . Telomeres, the complex structures at the ends of chromosomes are thought to be instrumental for both of these functions . However, recent insights in telomere biology suggest that these terminal structures do much more than just fulfill these two basic functions . Cytological data demonstrate that telomeres may play leading roles in chromatin organization and nuclear architecture during mitosis and meiosis . Moreover, non-functional telomeres may lead to genetic instability, a common prelude to cancer . Here, we review the basic functions of telomeres during chromosome replication and discuss the cytological aspects of telomere function during mitosis and meiosis. Riv Biol, 1998, 91(3), 425 - 57 Environmentally responsive mutator systems: toward a unifying perspective; Lieber MM; Biology has long sought a unifying principle . The behaviour of genetically controlled mutator processes adaptively responsive to stress may reflect such a principle . Related mutators exist in diverse organisms from bacteria to mammals . Such systems have evolved from one another and have defined the very evolution of organisms . Many of these mutator systems can determine in a developmental manner a ultramutability or hypermutability throughout the genome . Though the genetic control of high levels of mutability can reflect molecular features, such mutagenic processes reflect a deeper parameter involving forces and their configurations . These configurations must generate stable or uniform configurations from unstable ones throughout the genome and organism . Directed mutation becomes a generative process attuned to non-uniform forces of local niches and to the more uniform forces of a universal niche . The manner of mutagenic, attuned response depends on the level of genomic and transgenomic organization . This is reflected in hierarchies of evolution . Directed mutation is a feature of a universal, generative ordering process, and this feature is marked by a universal dimensionless constant . It is the dynamic consequence of such directed generation which ultimately confers adaptation through dynamic completion . This suggests an underlying, unitary, and necessary dynamics connecting ultramutability systems in all organisms and would serve, in complementation with a molecular approach, to elicit new and productive research avenues . One outcome would be the illustration of a unifying principle governing biological and physical phenomena. J Biol Chem, 1999 Apr 30, 274(18), 12222 - 8 Pre-steady-state reaction of 5-aminolevulinate synthase . Evidence for a rate-determining product release; Hunter GA et al.; 5-Aminolevulinate synthase (ALAS) is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and the alpha-subclass of purple bacteria . The pyridoxal 5'-phosphate cofactor at the active site undergoes changes in absorptive properties during substrate binding and catalysis that have allowed us to study the kinetics of these reactions spectroscopically . Rapid scanning stopped-flow experiments of murine erythroid 5-aminolevulinate synthase demonstrate that reaction with glycine plus succinyl-CoA results in a pre-steady-state burst of quinonoid intermediate formation . Thus, a step following binding of substrates and initial quinonoid intermediate formation is rate-determining . The steady-state spectrum of the enzyme is similar to that formed in the presence of 5-aminolevulinate, suggesting that release of this product limits the overall rate . Reaction of either glycine or 5-aminolevulinate with ALAS is slow (kf = 0.15 s-1) and approximates kcat . The rate constant for reaction with glycine is increased at least 90-fold in the presence of succinyl-CoA and most likely represents a slow conformational change of the enzyme that is accelerated by succinyl-CoA . The slow rate of reaction of 5-aminolevulinate with ALAS is 5-aminolevulinate-independent, suggesting that it also represents a slow isomerization of the enzyme . Reaction of succinyl-CoA with the enzyme-glycine complex to form a quinonoid intermediate is a biphasic process and may be irreversible . Taken together, the data suggest that turnover is limited by release of 5-aminolevulinate or a conformational change associated with 5-aminolevulinate release. J Med Chem, 1999 Apr 22, 42(8), 1459 - 65 Antineoplastic agents . 410 . Asymmetric hydroxylation of trans-combretastatin A-4; Pettit GR et al.; The South African willow tree Combretum caffrum has yielded a number of potent cancer cell growth inhibitors . The present SAR studies of the antineoplastic agent combretastatin A-4 (1c) were focused mainly on the olefinic bridge to determine the effects on cancer cell growth and, potentially, to better define the combretastatin A-4 binding site on tubulin . The geometric trans-isomer 3a of combretastatin A-4 was converted to the (1S,2S)- and (1R,2R)-vicinal diols 4c and 4d, respectively, under Sharpless' asymmetric dihydroxylation conditions . Cancer cell line testing showed the (1S, 2S)-diol 4c to be more potent than its enantiomer 4d . Diol 4c weakly inhibited tubulin polymerization (IC50 = 22 microM, versus 1.2 microM for combretastatin A-4), while 4d was inactive (IC50 > 40 microM) . Esterification of either stereoisomer at the diol and/or phenolic positions resulted in elimination of inhibitory activity. Aliment Pharmacol Ther, 1999 Mar, 13 Suppl 1, 13 - 8 Review article: the role of inflammation in the pathogenesis of gastric cancer; Ernst P; Helicobacter pylori induces infiltration of the gastric mucosa by polymorphonuclear cells and macrophages, as well as T and B lymphocytes . Paradoxically, this robust immune/inflammatory response cannot clear the infection, and thus leaves the host prone to complications resulting from chronic inflammation . One adverse consequence of this inflammatory response may be gastric cancer, as inflammation has been implicated in the development of intestinal metaplasia and mutations in oncogenes that precede the development of gastric adenocarcinoma . The gastric inflammatory response is affected somewhat, by the strain of H . pylori that infects the host . Thus, the more severe clinical manifestation associated with some strains may be attributed to the higher grade of inflammation that they induce . Both H . pylori and cytokines induced during infection can stimulate the recruitment and activation of inflammatory cells including neutrophils and macrophages . When activated, these cells produce inflammatory mediators that include reactive oxygen species (ROS) . These mediators impart an oxidative stress on the cells in the immediate vicinity, in this case, the gastric epithelium . Normally, oxidative stress is neutralized by natural antioxidants such as vitamin C, however, levels of this antioxidant in the gastric juice are decreased during infection . The increased levels of oxidants and decreased antioxidants create a stress that can change many processes in the gastric epithelium . For example, an accumulation of intracellular ROS regulates the expression of many genes and can induce DNA damage . Point mutations in the DNA that disrupt the expression and function of genes that inhibit cell growth (i.e . p53) are believed to contribute to the pathogenesis of gastric cancer . Several studies suggest that epithelial cell turnover is affected by the inflammatory response to H . pylori . This notion is supported by studies describing an increase in both epithelial cell proliferation, as well as cell death by apoptosis, in response to infection . Apoptosis is a regulated process of cell death that is triggered by H . pylori as well as various inflammatory mediators, including tumour necrosis factor and interferon-gamma . Activated T-cells also kill gastric epithelial cells directly . Moreover, the host response increases the expression of receptors for H . pylori and thus increases bacterial binding and the induction of apoptosis by the bacteria . There are several other immune/inflammatory responses that contribute to epithelial cell damage mucosa and the pathogenesis of gastric cancer . For example, gastric B cells produce autoreactive antibodies that bind to gastric epithelial cells . As a consequence of this antigen-antibody complex formation, complement becomes activated suggesting that some of the inflammation and epithelial cell damage is attributable to immune-complex formation . Epithelial cell death can then stimulate the proliferative response of epithelial cell precursors . In summary, the proposed model may explain how the gastric inflammatory response contributes to the pathogenesis of cancer . This model raises the possibility that it could be preferable to identify the patients at highest risk of developing gastric cancer and then apply an intervention that eliminates the infection and inflammatory response . Alternatively, clinical interventions should at least attenuate the oxidative stress that is directly attributed to inflammation . These mechanisms have to be examined in the paediatric population. Int Rev Cytol, 1999, 188, 203 - 55 Roles of reactive oxygen species: signaling and regulation of cellular functions; Gamaley IA et al.; Reactive oxygen species (ROS) are the side products (H2O2, O2.-, and OH.) of general metabolism and are also produced specifically by the NADPH oxidase system in most cell types . Cells have a very efficient antioxidant defense to counteract the toxic effect of ROS . The physiological significance of ROS is that ROS at low concentrations are able to mediate cellular functions through the same steps of intracellular signaling, which are activated by natural stimuli . Moreover, a variety of natural stimuli act through the intracellular formation of ROS that change the intracellular redox state (oxidation-reduction) . Thus, the redox state is a part of intracellular signaling . As such, ROS are now considered signal molecules at nontoxic concentrations . Progress has been achieved in studying the oxidative activation of gene transcription in animal cells and bacteria . Changes in the redox state of intracellular thiols are considered to be an important mechanism that regulates cell functions. Novartis Found Symp, 1999, 221, 152 - 63; discussion 163-6 pH homeostasis in acidophiles; Matin A; The acidophilic bacteria comprise an environmentally important group that includes pathogens . A fundamental requirement for existence in strongly acidic environments is generation of a large pH gradient (delta pH) to maintain the cytoplasmic pH near neutrality and safeguard the acid-labile cell constituents . These organisms require the capacity to extrude H+ across the thermodynamic barrier imposed by the delta pH . They also need special transport proteins that can function in strongly acidic environments . The quintessential device by which the acidophiles fulfil these requirements by generating a positive-inside membrane potential (delta psi) that mitigates both the force against which H+ must be extruded, as well as the influxing force of protons into the cells . The delta psi is generated through passive as well as active mechanisms . The former constitutes an H+ diffusion as well as a Donnan potential, and the latter involves electrogenic circulation of Cl-, and the operation of an electrogenic H+/K+ (Na+) antiporter. J Biol Chem, 1999 Apr 23, 274(17), 11995 - 2000 Adhesion of cultured bovine aortic endothelial cells to laminin-1 mediated by dystroglycan; Shimizu H et al.; Expression of dystroglycan (DG) by cultured bovine aortic endothelial (BAE) cells was confirmed by cDNA cloning from a BAE cDNA library, Northern blotting of mRNA, Western blotting of membrane proteins, and double immunostaining with antibodies against betaDG and platelet endothelial cell adhesion molecule-1 . Immunocytochemical analysis revealed localization of DG in multiple plaques on the basal side of resting cells . This patchy distribution was obscured in migrating cells, in which the most prominent staining was observed in the trailing edge anchoring the cells to the substratum . Biotin-labeled laminin-1 overlay assay of dissociated BAE membrane proteins indicated the interaction of laminin-1 with alphaDG . The laminin alpha5 globular domain fragment expressed in bacteria and labeled with biotin could also bind alphaDG on the membrane blot, and the unlabeled fragment disrupted the binding of biotin-laminin-1 to alphaDG . The interaction of biotin-laminin-1 with alphaDG was inhibited by soluble alphaDG contained in the conditioned medium from DG cDNA-transfected BAE cells and by a series of glycosaminoglycans (heparin, dextran sulfate, and fucoidan) . Soluble alphaDG in the conditioned medium inhibited the adhesion of BAE cells to laminin-1-coated dishes, whereas it had no effect on their adhesion to fibronectin . All three glycosaminoglycans that disrupted the biotin-laminin-1 binding to alphaDG inhibited BAE cell adhesion to laminin-1, whereas they failed to inhibit the adhesion to fibronectin . These results indicate a role of DG as a non-integrin laminin receptor involved in vascular endothelial cell adhesion to the extracellular matrix. Int J Tuberc Lung Dis, 1999 Apr, 3(4), 354 - 7 Application of a computer-directed automated microscope in mycobacteriology; Somoskovi A et al.; Microscopy is currently the fastest, cheapest and most easily performed technique in mycobacteriology; it can be used in any laboratory . However, the sensitivity of microscopy is unsatisfactory and it is time-consuming . To eliminate these drawbacks, we have constructed a computer-directed automated microscope . To evaluate the equipment, we examined a total of 132 smears of sputum and 74 smears of liquid media . Manual microscopy was positive for 53 and negative for 79 sputum smears, while automated microscopy was positive for 55 and negative for 77 sputum smears . Both methods furnished 50 positive and 24 negative smears of liquid media . We conclude that the automated microscope is able to detect acid-fast bacteria, the examination procedure with the instrument is more rapid (1.8-3.5 min/slide) and it is always possible to follow the standard recommendations of microscopy. Vet Immunol Immunopathol, 1999 Mar 1, 67(4), 327 - 40 Production and functional characterization of recombinant bovine interleukin-8 as a specific neutrophil activator and chemoattractant; Caswell JL et al.; Interleukin-8, a member of the chemokine family of cytokines, is a potent neutrophil chemoattractant in many non-rodent species . In this study, recombinant bovine interleukin-8 (rbIL-8) was expressed in bacteria as a glutathione-S-transferase fusion protein . The fusion protein was purified by glutathione-Sepharose affinity chromatography and recombinant rbIL-8 was eluted by cleaving with thrombin . The purified rbIL-8 molecule was approximately 8 kDa and was confirmed as authentic IL-8 by Western analysis . Recombinant bovine IL-8 induced specific dose-dependent in vitro chemotaxis of neutrophils at doses as low as 1.0 ng/ml, and this activity was inhibited by pre-treatment of rbIL-8 with a monoclonal antibody to ovine IL-8 . Neutrophils exposed to rbIL-8 developed pseudopodia and became elongated as determined by microscopic analysis and flow cytometry . Injection of 3.3 ng to 3.3 microg of rbIL-8 into the skin of a normal calf induced dose-dependent recruitment of neutrophils but not eosinophils . Intravascular margination of neutrophils was obvious at the injection sites from 15 to 60 min after administration of rbIL-8, and extravascular neutrophil numbers increased steadily from 1 to 18 h after injection . Neutrophils with morphologic features of apoptosis were detected in these lesions at 18 and 30 h after injection, and this correlated with reduction in the number of dermal neutrophils . These results confirm unequivocally that bovine IL-8 functions as a neutrophil, but not an eosinophil, chemoattractant in vitro and in vivo. Farmaco, 1998 Oct-Nov, 53(10-11), 709 - 17 Synthesis and activity of cephalosporins containing an oxyiminomethylene functionality in the ortho-position of a phenyl- or phenoxyacetic acid C-7 side chain substituent; Albanese D et al.; A new series of cephalosporins having in the C-7 side chain a phenyl- or a phenoxyacetamido group bearing an oxyiminomethyl function in the ortho-position of the aromatic ring was prepared . Their in vitro activity was tested against both Gram+ and Gram- strains. Prof Nurse, 1999 Feb, 14(5), 329 - 33 Larval therapy in wound debridement; Rayner K; The use of larvae (maggots) in wound management was popular in the 1930s . Now the advent of multi-resistant strains of bacteria has led to its reintroduction in some hospitals, when other avenues have been exhausted. Eur J Med Res, 1999 Apr 27, 4(4), 161 - 4 Familial Mediterranean fever . No role of Mycobacterium tuberculosis in ten patients; Akcan Y et al.; BACKGROUND: Tuberculosis (TB) and Familial mediterranean fever (FMF) are two common diseases in our region, Turkey . Both share some properties in common: Both cause AA type amyloidosis and have association with some immunological abnormalities . Upon incidentally observing Mycobacterium tuberculosis in bone marrow biopsies of three patients with FMF in a previous study, we intended to elucidate this association prospectively . MATERIAL AND METHODS: In this study, we examined prospectively 10 FMF patients, 5 male and 5 female, with a median duration of 31 years disease activity . All were under colchicine therapy . They had no sign of renal involvement . The bone marrow biopsies of these patients were examined for the presence of M . tuberculosis by Polymerase chain reaction (PCR), BACTEC culture and pathological stains . Pathological examination was performed for the existence of granuloma and amyloid deposition by hematoxylin-eosin, Crystal Violet and Congo red stains . RESULTS: The examination of all bone marrow specimens by the mentioned methods suggest that Mycobacterium tuberculosis has no role in the ethiopathogenesis of FMF . Although the patients had a positive family history of 60% for tuberculosis and in 80% of them with positive tuberculin skin test . CONCLUSIONS: We concluded that although there seemed to be a kind of association between both diseases, this relationship is not via the direct existence of bacteria itself . Considering high family history and skin test positivity, one should look for the presence of autoimmune mechanisms under this suspicious relationship between tuberculosis and FMF . Also, this is the first study examined the state of amyloidosis in the bone marrow at an earlier stage of FMF without overt renal findings. Gut, 1999 May, 44(5), 643 - 52 Mast cells are an important cellular source of tumour necrosis factor alpha in human intestinal tissue; Bischoff SC et al.; BACKGROUND: Several inflammatory disorders of the intestine are characterised by enhanced expression of tumour necrosis factor alpha (TNF-alpha) . Monocytes and macrophages have been suggested as a major cellular source of TNF-alpha in human gut, whereas mast cells, although known to be capable of producing TNF-alpha, have been poorly examined in this respect . AIMS: To investigate whether human intestinal mast cells can produce TNF-alpha, and which factors regulate TNF-alpha production in these cells . METHODS: Mast cells were isolated from surgery tissue specimens of patients undergoing bowel resection because of cancer . Immunohistochemical studies were performed in biopsy specimens derived from 13 patients (two healthy controls, four with Crohn's disease, four with ulcerative colitis, three others) . TNF-alpha mRNA and protein expression were studied in vitro by polymerase chain reaction, RNAse protection assay, western blot, and enzyme linked immunosorbent assay in isolated purified human intestinal mast cells stimulated by IgE receptor crosslinking, intestinal bacteria, and lipopolysaccharide . Cellular localisation of TNF-alpha was examined by immunohistochemistry . RESULTS: TNF-alpha mRNA and protein were expressed constitutively in isolated human intestinal mast cells . Expression of TNF-alpha mRNA and release of TNF-alpha protein were substantially enhanced by IgE receptor crosslinking and by coculture of mast cells with intestinal bacteria; lipopolysaccharide had only marginal effects . Immunohistochemical studies revealed that approximately 60% of the lamina propria cells with immunoreactivity for TNF-alpha were mast cells . CONCLUSIONS: The data show that mast cells are an important source of TNF-alpha in the human intestinal mucosa. EMBO J, 1999 Apr 15, 18(8), 2284 - 93 Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome; Suppmann S et al.; The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo . A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10% . This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS . When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced . When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression . The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present . The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs. Oral Microbiol Immunol, 1999 Feb, 14(1), 49 - 55 Adherence of Peptostreptococcus micros morphotypes to epithelial cells in vitro; Kremer BH et al.; Peptostreptococcus micros, which is associated with oral and non-oral mixed anaerobic infections, occurs in three colony morphotypes, the smooth type, the rough type and the smooth variant of the rough type . These types differ in surface structures; the rough type expresses large fibrillar surface appendages, which are absent on the surface of both the smooth and the smooth variant of the rough type . To determine the role of these surface structures in adherence we characterized the adherence of the three morphotypes of P . micros to epithelial cells in vitro . Although all three types adhered well to epithelial cells, adhering numbers of the rough type were significantly lower than those of the smooth and the smooth variant of the rough type . Protease treatment increased the adherence of the rough type of the level of the two other types . The adherence of all three types was reduced more than 85% by treatment with 10 mM sodium periodate . Furthermore, the adherence was pH independent and could not be blocked by incubation with antisera to the bacteria . In addition, we determined the capacity to invade epithelial cells by P . micros . In an acridine orange assay such invasion could not be detected . Our results suggest that the adherence of P . micros to epithelial cells is mediated by periodate-sensitive extracellular polysaccharides and that the protruding fibril-like protein surface structures of the rough type have an obstructive effect on the adherence. J Clin Microbiol, 1999 May, 37(5), 1426 - 30 Distribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patients; Amano A et al.; Fimbriae (FimA) of Porphyromonas gingivalis are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues . We previously demonstrated that fimA can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the fimA gene . In the present study, we attempted to detect the four different fimA genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method . Four sets of fimA type-specific primers were designed for the PCR assay . These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the fimA gene . Positive PCR results were observed with reference strains of P . gingivalis in a type-specific manner . All other laboratory strains of oral and nonoral bacteria gave negative results . The sensitivity of the PCR assay for fimA type-specific detection was between 5 and 50 cells of P . gingivalis . Clinical samples were obtained from saliva and subgingival plaque from deep pockets (>/=4 mm) of 93 patients with periodontitis . Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR . The presence of P . gingivalis was demonstrated in 73 patients (78.5%), and a single fimA gene was detected in most patients . The distribution of the four fimA types among the P . gingivalis-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6 . 8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8% . P . gingivalis with type II fimA was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (>/=8 mm) pockets . These results suggest that P . gingivalis strains that possess type II fimA are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases. J Clin Microbiol, 1999 May, 37(5), 1254 - 9 Prevalence of Mycobacterium avium in slaughter pigs in The Netherlands and comparison of IS1245 restriction fragment length polymorphism patterns of porcine and human isolates; Komijn RE et al.; A significant increase in the incidence of caseous lesions in the lymph nodes of slaughter pigs prompted a large-scale investigation in five slaughterhouses in The Netherlands . In total, 158,763 pigs from 2,899 groups underwent gross examination . At least one pig with caseous lesions in the submaxillary and/or mesenteric lymph nodes was observed in each of 154 of the 2,899 groups examined (5%) . In total, 856 pigs (0.5%) were affected . As many as five pigs in each of 141 of the 154 positive groups (91.5%) had lymph node lesions . Greater numbers of pigs with affected lymph nodes were found in 13 groups (8.5%) . Four pigs had lesions in the kidneys, liver, or spleen . Acid-fast bacteria were detected by microscopic examination of 121 of 292 Ziehl-Neelsen-stained smears of caseous lesions (41%) . In a follow-up study, Mycobacterium avium complex (MAC) bacteria were isolated from 219 of 402 affected lymph nodes (54.2%) . Ninety-one of the isolated strains were analyzed by restriction fragment length polymorphism (RFLP) typing with insertion sequence IS1245 as a probe . All but 1 of these 91 strains contained IS1245 DNA, indicating that pigs in The Netherlands carried almost exclusively M . avium bacteria and no other bacteria of MAC . Only one pig isolate exhibited the bird-type RFLP pattern . MAC isolates from 191 human patients in The Netherlands in 1996 were also typed by RFLP analysis . Computer-assisted analysis showed that the RFLP patterns of 61% of the human isolates and 59% of the porcine isolates were at least 75% similar to the RFLP patterns of the other group of strains . This indicates that pigs may be an important vehicle for M . avium infections in humans or that pigs and humans share common sources of infection. Can J Gastroenterol, 1999 Mar, 13(2), 147 - 51 Changing parenteral nutrition administration sets every 24 h versus every 48 h in newborn infants; Fox M et al.; OBJECTIVE: To determine whether changing total parenteral nutrition fluid administration sets (TAS) every 48 h rather than every 24 h results in a greater infusate contamination rate . PATIENTS AND METHODS: Prospectively, 166 infants were assigned at random to have TAS changed either every 24 h or every 48 h . Samples of the infusate were cultured to determine contamination rates of the infusate in the sets and were tested from 149 of these infants . TAS was replaced every 24 h in the control group, and 445 amino acid plus dextrose solutions (AADS) and 449 lipid emulsions samples were taken for bacterial culture . Fungal cultures were also performed on 449 samples . The study group had TAS replaced every 48 h, and 454 samples of AADS were cultured for bacteria . The numbers of lipid emulsion samples sent for bacterial culture and fungal culture were 449 and 440, respectively . Information on type of intravenous access device, administration of antibiotics and blood cultures was also collected . RESULTS: There was no difference in bacterial contamination rates for AADS or lipid emulsion from TAS changed every 24 or 48 h (c2, P>0.05) . Lipid emulsion sampled from the 24 h group showed a statistically significant higher rate of fungal contamination than specimens from the 48 h group (P<0.01) . CONCLUSIONS: Changing TAS every 48 h versus 24 h does not increase the contamination rate of infusate in newborns. Vet Clin North Am Small Anim Pract, 1999 Mar, 29(2), 471 - 500, vi-vii Gastrointestinal immunity in health and disease; Elwood CM et al.; The gastrointestinal lymphoid tissue (GALT) is under constant antigenic challenge from bacteria and food and must be able to distinguish between benign and pathogenic organisms . Recent advances in understanding the organization and function of GALT reveal how it is able to direct appropriate immune responses according to the nature of the antigen and how inappropriate immune responses can lead to local and systemic immunopathology and/or infection . The interaction of the normal bowel flora and GALT is critical to normal local and systemic immune function and plays a major role in the pathogenesis of some immune-related diseases . This review draws upon information from veterinary, human, and laboratory animal studies to provide an update of mechanisms and consequences of function and dysfunction in the gastrointestinal immune system. Bioorg Med Chem Lett, 1999 Mar 8, 9(5), 781 - 6 Synthesis and electrochemical study of a new chiral tris-catecholamide analogue of enterobactin; Cheraiti N et al.; The comparison of siderophore complex redox potentials with those of physiological reductants may aid in the clarification of the mechanism of iron metabolism . In this paper, a new chiral tris-catecholamide compound N,N',N''-tris-(2,3-dihydroxybenzoyl)-1,1,1-tris-(L-methioninemethyl++ +)-ethane or H6L (11) has been synthesised in nine steps, and may mimic the release of iron from enterobactin to the agents which are directly involved in cell metabolism . The choice of methionine as a constituent of the siderophore incorporates divalent sulphur which leads to the increase of the reduction potential of the siderophore, and consequently facilitates the iron release {Fe(III)/Fe(II) redox potential E(1/2)=-0.749 V vs (SCE)}. Mol Microbiol, 1999 Mar, 31(5), 1295 - 305 Transcription initiation in Archaea: facts, factors and future aspects; Soppa J; The basal apparatus for transcription initiation in Archaea is more closely related to the eukaryal than to the bacterial counterpart . The understanding of archaeal transcription initiation has been deepened by recent advances, which include genome sequencing, biochemical approaches and the structure determination of a protein DNA complex . Archaeal promoter elements, transcription factors, RNA polymerase and their interactions are discussed and compared with the eukaryal situation . It is emerging that transcription initiation is not uniform in Archaea . A minimal set of promoter elements and transcription factors is conserved, but the relative importance for transcription initiation can vary . Furthermore, additional basal transcription factors and promoter elements seem to be crucial in subgroups of Archaea . Finally, some aspects of global as well as gene-specific transcriptional regulation are discussed. Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4615 - 20 A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane; Vannelli T et al.; Methylobacterium sp . strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source . Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis . The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments . The sequences of these fragments, which extended over more than 17 kb, were determined . Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate . The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB . Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea . In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens . The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium . Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism. Proc Natl Acad Sci U S A, 1999 Apr 13, 96(8), 4348 - 53 The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer; Myllykallio H et al.; Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction . Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc . These electron carriers participate in both respiratory and photosynthetic electron-transfer chains . On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs . Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined . It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R . capsulatus counterpart, Cyt cyRc . Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme . It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions . These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains? RNA, 1999 Apr, 5(4), 562 - 73 Nonsense mutations in the alcohol dehydrogenase gene of Drosophila melanogaster correlate with an abnormal 3' end processing of the corresponding pre-mRNA; Brogna S; From bacteria to mammals, mutations that generate premature termination codons have been shown to result in the reduction in the abundance of the corresponding mRNA . In mammalian cells, more often than not, the reduction happens while the RNA is still associated with the nucleus . Here, it is reported that mutations in the alcohol dehydrogenase gene (Adh) of Drosophila melanogaster that generate premature termination codons lead to reduced levels of cytoplasmic and nuclear mRNA . Unexpectedly, it has been found that the poly(A) tails of Adh mRNAs and pre-mRNAs that carry a premature termination codon are longer than in the wild-type transcript . The more 5' terminal the mutation is, the longer is the poly(A) tail of the transcript . These findings suggest that the integrity of the coding region may be required for accurate mRNA 3' end processing. Cell, 1999 Apr 2, 97(1), 75 - 84 Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism; Velankar SS et al.; We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway . One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex . The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex . In both complexes, the protein is monomeric . Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor . Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism. Probl Tuberk, 1999, (1), 30 - 1 {Results of polyresistant tuberculosis treatment: data of the Santarishkes Republican tuberculous hospital}; Mishkinis K et al.; In 1994-1996 the Santarishkes republican tuberculous hospital (Lithuania) has admitted 98 patients (82 males, 16 females) with polyresistant tuberculosis (M . tuberculosis were resistant to at least isoniazid and rifampicin) . Of these, 13 patients were untreated, 17 had recurrences, 68 had chronic tuberculosis . After polyresistance of M . tuberculosis was found, the patients received an individual treatment with sensitive drugs . 20 patients were operated . The absence of bacteria was achieved only in 24 (24.5%) patients . Tuberculous lesions in the lungs disappeared and clinical symptoms relieved in 14 of them . In 74(75.5%) nonresponders the course of the disease was unfavourable . Negative treatment outcomes were observed in 38.5%, 64.7% and 85.3% of new-onset, recurrent and chronic cases, respectively. Probl Tuberk, 1999, (1), 12 - 3 {Analysis of tuberculosis morbidity in the southeastern district of Moscow}; Kovaleva SI et al.; The present-day poor epidemiological conditions are marked by an increase in tuberculosis detection rates (1.43%) with low coverage (28.0%) of the population with preventive fluorographic surveys and a high proportion (39.0%) of young patients (aged 18-39 years) among new cases . The shares of advanced and acute forms of tuberculosis were 7.2 and 11.4%, respectively . A decay phase was detected in 50.6%, 52.6% of patients isolated bacteria . In children, tuberculosis morbidity increased by 28.6% mainly among the unregistered extrafamilial contacts . Most patients (75.8%) detected upon their referral to the polyclinics and general hospitals had a high proportion of extrapulmonary tuberculosis . Thus, the above trends of morbidity show its high potential level. Br J Gen Pract, 1998 Nov, 48(436), 1751 - 4 Why patients consult when they cough: a comparison of consulting and non-consulting patients; Cornford CS; BACKGROUND: Although it is the commonest symptom presented to general practitioners (GPs), little is known about why someone decides to consult with a cough . AIM: To describe the illness behaviour of patients with a cough . METHOD: Patients who had consulted a GP because of a cough, and a group of subjects who had recently had a cough but had not consulted, were interviewed in a qualitative study that investigated how they made sense of their illness . RESULTS: Consulting patients understood their cough to be abnormally severe, whereas non-consulting subjects regarded their cough as 'normal' and mild . Consulting patients thought the cough would interfere with social roles and non-consulting subjects did not . The consulting patients were much more likely to be worried about the cough than the non-consulting subjects . In particular, half of the consulting patients were worried about their hearts, whereas the non-consulting subjects were not . The two groups did not distinguish bacteria from viruses, and did not differ in beliefs about the role of antibiotics that they thought were needed for severe coughs . Both groups had concerns about pollution . CONCLUSIONS: For consulting patients, cough breached the taken for granted property' of health that the non-consulting subjects with a cough were able to maintain . Cough, for the consulting patients, was not a trivial illness. Am J Physiol, 1999 Apr, 276(4 Pt 1), L650 - 8 Effects of endotoxin on surfactant protein A and D stimulation of NO production by alveolar macrophages; Wright JR et al.; Surfactant protein (SP) A and SP-D affect numerous functions of immune cells including enhancing phagocytosis of bacteria and production of reactive species . Previous studies have shown that SP-A and SP-D bind to a variety of bacteria and to the lipopolysaccharide (LPS) components of their cell walls . In addition, purified preparations of SPs often contain endotoxin . The goals of this study were 1) to evaluate the effects of SP-A and SP-D and complexes of SPs and LPS on the production of nitric oxide metabolites by rat alveolar macrophages and 2) to evaluate methods for the removal of endotoxin with optimal recovery of SP . Incubation of SP-A or SP-D with polymyxin, 100 mM N-octyl-beta-D-glucopyranoside, and 2 mM EDTA followed by dialysis was the most effective method of those tested for reducing endotoxin levels . Commonly used storage buffers for SP-D, but not for SP-A, inhibited the detection of endotoxin . There was a correlation between the endotoxin content of the SP-A and SP-D preparations and their ability to stimulate production of nitrite by alveolar macrophages . SP-A and SP-D treated as described above to remove endotoxin did not stimulate nitrite production . These studies suggest that the functions of SP-A and SP-D are affected by endotoxin and illustrate the importance of monitoring SP preparations for endotoxin contamination. Genomics, 1999 Apr 15, 57(2), 306 - 9 A 1.5-Mb contig within the cat eye syndrome critical region at human chromosome 22q11.2; Johnson A et al.; We have constructed a 1.5-Mb contig spanning the distal half of the critical region for cat eye syndrome on human chromosome 22 from D22S543 to D22S181 . The contig consists of 20 P1 artificial chromosome (PAC) clones and 11 bacterial artificial chromosome (BAC) clones screened from 2 BAC and 2 PAC libraries . Continuous overlap between the clones was confirmed using vectorette PCR and riboprobes . Despite the instability of this region in a previous YAC contig, only 1 BAC showed a minor instability and then in only one isolation . This contig is now providing the basis for genomic sequencing and gene identification in the cat eye syndrome critical region . Plant Physiol, 1999 Apr, 119(4), 1483 - 96 Oxidative turnover of soybean root glutamine synthetase . In vitro and in vivo studies Ortega JL, Roche D, Sengupta-Gopalan C. Glutamine synthetase (GS) is the key enzyme in ammonia assimilation and catalyzes the ATP-dependent condensation of NH3 with glutamate to produce glutamine . GS in plants is an octameric enzyme . Recent work from our laboratory suggests that GS activity in plants may be regulated at the level of protein turnover (S.J . Temple, T.J . Knight, P.J . Unkefer, C . Sengupta-Gopalan {1993} Mol Gen Genet 236: 315-325; S.J . Temple, S . Kunjibettu, D . Roche, C . Sengupta-Gopalan {1996} Plant Physiol 112: 1723-1733; S.J . Temple, C . Sengupta-Gopalan {1997} In C.H . Foyer, W.P . Quick, eds, A Molecular Approach to Primary Metabolism in Higher Plants . Taylor & Francis, London, pp 155-177) . Oxidative modification of GS has been implicated as the first step in the turnover of GS in bacteria . By incubating soybean (Glycine max) root extract enriched in GS in a metal-catalyzed oxidation system to produce the.OH radical, we have shown that GS is oxidized and that oxidized GS is inactive and more susceptible to degradation than nonoxidized GS . Histidine and cysteine protect GS from metal-catalyzed inactivation, indicating that oxidation modifies the GS active site and that cysteine and histidine residues are the site of modification . Similarly, ATP and particularly ATP/glutamate give the enzyme the greatest protection against oxidative inactivation . The roots of plants fed ammonium nitrate showed a 3-fold increase in the level of GS polypeptides and activity compared with plants not fed ammonium nitrate but without a corresponding increase in the GS transcript level . This would suggest either translational or posttranslational control of GS levels. J Bacteriol, 1999 Apr, 181(8), 2655 - 8 Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation; Michel-Reydellet N et al.; We herein report that Azorhizobium caulinodans PII and GlnK are not necessary for glutamine synthetase (GS) adenylylation whereas both proteins are required for complete GS deadenylylation . The disruption of both glnB and glnK resulted in a high level of GS adenylylation under the condition of nitrogen fixation, leading to ammonium excretion in the free-living state . PII and GlnK also controlled nif gene expression because NifA activated nifH transcription and nitrogenase activity was derepressed in glnB glnK double mutants, but not in wild-type bacteria, grown in the presence of ammonia. J Bacteriol, 1999 Apr, 181(8), 2430 - 9 The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle; Reisenauer A et al.; In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle . To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene . Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle . PfliQ is activated earlier than PccrM . Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ . This difference in affinity correlates with temporal changes in the cellular levels of CtrA . Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression . Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes . However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes . We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle. Lett Appl Microbiol, 1999 Mar, 28(3), 211 - 5 Growth of moulds inoculated into commercial mineral water; Fujikawa H et al.; The growth of mould spores of Penicillium sp . and Cladosporium sp . inoculated in a commercial mineral water product was studied . The strains had been isolated as fungal foreign bodies in commercial mineral waters . In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage . In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies . The viable colony counts and the beta-glucans concentration in the samples increased during storage . These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies. C R Acad Sci III, 1999 Feb-Mar, 322(2-3), 167 - 75 Adaptive response and induced resistance; Joiner MC et al.; Cellular stress responses are upregulated following exposure to radiation and other DNA-damaging agents . Therefore radiation response can be dose dependent so that small acute exposures (and possibly exposures at very low dose rates?) are more lethal per unit dose than larger exposures above a threshold (typically 10-40 cGy) where induced radioprotection is triggered . We have termed these interlinked phenomena low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases . HRS/IRR has been recorded in cell-survival studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal normal-tissue models in vivo . There is indirect evidence that cell survival-related HRS/IRR in response to single doses is a manifestation of the same underlying mechanism that determines the well-known adaptive response in the two-dose case and that it can be triggered by high- and low-LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents . Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis . Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified . Net cancer risk is a balance between cell transformation and cell kill . Our known low-dose cell-survival responses suggest that lethality may more than compensate for transformation at low radiation doses . However, adaptive reduction in sensitivity to radio-mutation has also been reported, which implies the existence also of enhanced mutation following very low single doses . So far this has not been confirmed, but provided the trigger dose for mutational protection was lower than the trigger dose for protection against cytotoxicity, cell killing would still dominate over at least the first 10 cGy of low-LET exposure . This would lead to a non-linear, threshold, dose-risk relationship and even provide some explanation for anecdotal reports of apparent 'health promoting' effects and lowered cancer risk from very low exposure to ionising radiation. C R Acad Sci III, 1999 Feb-Mar, 322(2-3), 143 - 9 Mechanisms of mutagenesis in mammalian cells . Application to human thyroid tumours; Sarasin A et al.; Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements . These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway . The molecular process leading to mutagenesis depends largely on the type of DNA lesions . Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions . The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors . Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone . These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell . In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process . The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells . IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals . The breakage of the double-helix is a difficult lesion to repair . Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements . Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor . The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis . In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes . These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5 . The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84% . This frequency was only 15% in 'spontaneous' PTC . The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different . Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones . The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%) . The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test) . In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present . A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation . The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one . It will be interesting to use this time lag in accidental or therapeutic-irradiated p J Virol, 1999 May, 73(5), 3810 - 7 A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection; Bruni R et al.; Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0) . ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells . We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22 . This protein (apparent Mr of 60,000) has not been previously described and has no known motifs . Analyses of p60 revealed the following . (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R . Bruni and B . Roizman, J . Virol . 72:8525-8531, 1998) bound all forms of ICP22 . The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms . (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22 . (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm . In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed . Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase . (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei . Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0 . Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr . The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases . The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22. Protein Eng, 1999 Feb, 12(2), 155 - 62 Mutation of a highly conserved aspartate residue in subdomain IX abolishes Fer protein-tyrosine kinase activity; Cole LA et al.; Before the structure of cAMP-dependent protein kinase had been solved, sequence alignments had already suggested that several highly conserved peptide motifs described as kinase subdomains I through XI might play some functional role in catalysis . Crystal structures of several members of the protein kinase superfamily have suggested that the nearly invariant aspartate residue within subdomain IX contributes to the conformational stability of the catalytic loop by forming hydrogen bonds with backbone amides within subdomain VI . However, substitution of this aspartate with alanine or threonine in some protein kinases have indicated that these interactions are not essential for activity . In contrast, we show here that conversion of this aspartate to arginine abolished the catalytic activity of the Fer protein-tyrosine kinase when expressed either in mammalian cells or in bacteria . Structural modeling predicted that the catalytic loop of the FerD743R mutant was disrupted by van der Waal's repulsion between the side chains of the substituted arginine residue in subdomain IX and histidine-683 in subdomain VI . The FerD743R mutant model predicted a shift in the peptide backbone of the catalytic loop, and an outward rotation of histidine-683 and arginine-684 side chains . However, the position and orientation of the presumptive catalytic base, aspartate-685, was not substantially changed . The proposed model explains how substitutions of some, but not all residues could be tolerated at this nearly invariant aspartate in kinase subdomain IX. Math Biosci, 1999 Mar 15, 157(1-2), 217 - 36 Resistance of a food chain to invasion by a top predator; Kooi BW et al.; We study the invasion of a top predator into a food chain in a chemostat . For each trophic level, a bioenergetic model is used in which maintenance and energy reserves are taken into account . Bifurcation analysis is performed on the set of nonlinear ordinary differential equations which describe the dynamic behaviour of the food chain . In this paper, we analyse how the ability of a top predator to invade the food chain depends on the values