Biochemistry, 1996 Nov 19, 35(46), 14486 - 502 A model for the photosystem II reaction center core including the structure of the primary donor P680; Svensson B et al.; For a detailed understanding of the function of photosystem II (PSII), a molecular structure is needed . The crystal structure has not yet been determined, but the PSII reaction center proteins D1 and D2 show homology with the L and M subunits of the photosynthetic reaction center from purple bacteria . We have modeled important parts of the D1 and D2 proteins on the basis of the crystallographic structure of the reaction center from Rhodopseudomonas viridis . The model contains the central core of the PSII reaction center, including the protein regions for the transmembrane helices B, C, D, and E and loops B-C and C-D connecting the helices . In the model, four chlorophylls, two pheophytins, and the nonheme Fe2+ ion are included . We have applied techniques from computational chemistry that incorporate statistical data on side-chain rotameric states from known protein structure and that describe interactions within the model using an empirical potential energy function . The conformation of chlorophyll pigments in the model was optimized by using exciton interaction calculations in combination with potential energy calculations to find a solution that agrees with experimentally determined exciton interaction energies . The model is analyzed and compared with experimental results for the regions of P680, the redox active pheophytin, the acceptor side Fe2+, and the tyrosyl radicals TyrD and TyrZ . P680 is proposed to be a weakly coupled chlorophyll a pair which makes three hydrogen bonds with residues on the D1 and D2 proteins . In the model the redox-active pheophytin is hydrogen bonded to D1-Glu130 and possibly also to D1-Tyr126 and D1-Tyr147 . TyrD is hydrogen bonded to D2-His190 and also interacts with D2-Gln165 . TyrZ is bound in a hydrophilic environment which is partially constituted by D1-Gln165, D1-Asp170, D1-Glu189, and D1-His190 . These polar residues are most likely involved in proton transfer from oxidized TyrZ or in metal binding. J Mol Biol, 1996 Nov 15, 263(5), 627 - 36 Selective methyl group protonation of perdeuterated proteins; Rosen MK et al.; Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy . One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins . In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein . We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples . Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids . In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated . This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis . As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR. Blood, 1996 Nov 15, 88(10), 3755 - 64 Role of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in the development of an acute neutrophil inflammatory response in mice; Metcalf D et al.; The intraperitoneal injection into mice of casein preparations containing bacteria induced a rapid accumulation of neutrophils within 3 hours due to selective release of mature cells from the bone marrow . Significant increases in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurred in the peritoneal cavity during the process, but the intraperitoneal injection of neither CSF induced a significant accumulation of neutrophils and the coinjection of G-CSF and casein failed to enhance the neutrophil response . The lack of involvement of either CSF in the neutrophil migration was confirmed by the development of typical neutrophil exudates when casein was injected into mice with inactivation of the genes encoding GM-CSF, G-CSF, or the beta-common chain of the GM-CSF receptor . However, preinjection of G-CSF increased the number of marrow neutrophils available for migration and did result in increased numbers of neutrophils in the peritoneal cavity after casein injection . Typical eosinophil inflammatory responses to the injection of casein or thioglycollate occurred in GM-CSF -/ -mice but not in beta c -/- mice, suggesting that interleukin-5 was necessary for this response. Cancer Res, 1996 Nov 15, 56(22), 5186 - 91 Guanylyl cyclase C is up-regulated by nonparenchymal cells and hepatocytes in regenerating rat liver; Scheving LA et al.; Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin produced by bacteria as well as for the newly discovered mammalian hormones guanylin and uroguanylin . Ligand activation of GC-C causes it to produce cyclic GMP inside target cells . Although once thought to be restricted to the intestine, GC-C mRNA has recently been detected in other tissues . We now examine the expression, localization, and activation of this glycoprotein after partial hepatectomy in rats . By immunoblot analysis, GC-C protein appeared as early as 4 h after partial hepatectomy, reached its maximal expression (a 30-fold increase) between 24 and 48 h, and returned to low baseline levels at 96 h . During the regenerative period, we detected two GC-C isoforms that differed in their size, temporal expression, and carbohydrase sensitivities . We showed that 131- and 140-kDa GC-C isoforms represented immature and mature GC-C glycoforms on the basis of endoglycosidase H and PNGase sensitivities . Cell separation experiments revealed that the nonparenchymal cell fractions of regenerating liver contained four times as much GC-C as purified hepatocytes . Immunohistochemistry confirmed these findings . The exuberant expression of GC-C by nonparenchymal cells and, to a lesser extent, hepatocytes suggests a role for cyclic GMP in liver regeneration. J Immunol, 1996 Nov 15, 157(10), 4568 - 75 Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages; Schlesinger LS et al.; Phagocytosis of the virulent Erdman and H37Rv strains of Mycobacterium tuberculosis, but not that of the attenuated H37Ra strain, by human macrophages is mediated by the mannose receptor (MR) in addition to complement receptors . We have recently determined that a major capsular lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serves as a ligand for the MR during phagocytosis of bacteria . In this study we directly compare uptake of Erdman, H37Rv, and H37Ra LAM by human macrophages and assess the relative contribution of the MR in this process . Microspheres coated with LAM served as model phagocytic particles for studies of LAM as a capsular ligand . Uptake (37 degrees C) of LAM microspheres by monocyte-derived macrophages was greatest for Erdman LAM and intermediate for H37Rv and H37Ra LAM compared with that of buffer microspheres or microspheres coated with LAM from a nontuberculosis strain of mycobacterium (AraLAM) . Inhibition of microsphere uptake in the presence of mannan or mannose-BSA was highest for Erdman LAM (75 +/- 8 and 50 +/- 7%, respectively) and H37Rv LAM (57 +/- 13 and 21 +/- 5%, respectively) relative to H37Ra LAM (36 +/- 16 and 22 +/- 11 %, respectively) . Inhibition of microsphere uptake in the presence of anti-MR Ab followed a similar pattern: Erdman LAM (80 +/- 9%) > H37Rv LAM (53 +/- 1%) > H37Ra LAM (26 +/- 12%) . Attachment (4 degrees C) of microspheres coated with Erdman LAM, H37Rv LAM, and H37Ra LAM was enhanced 12-, 5-, and 4-fold, respectively, compared with that of microspheres coated with AraLAM, and mannose-BSA inhibited attachment of these microspheres by 82 +/- 7, 69 +/- 8, and 12 +/- 17% . Galactose-BSA did not inhibit attachment of any LAM microsphere groups . Chromatographic analyses of mild acid hydrolysates of LAM from Erdman, H37Rv, and H37Ra all revealed the major terminal dimannosyl units . These studies demonstrate differences in the ability of LAM from different M . tuberculosis strains to mediate adherence to macrophages and to serve as ligands for the macrophage MR despite the presence of terminal dimannosyl units . Thus, these studies point toward other subtle structural alterations in LAM among strains that influence initial interactions with human phagocytes. J Immunol Methods, 1996 Nov 13, 198(2), 187 - 98 Mapping of epitopes recognized by PM/Scl autoantibodies with gene-fragment phage display libraries; Bluthner M et al.; Sera from patients suffering from the polymyositis/scleroderma overlap syndrome (PM/Scl) recognize two antigenically non-related proteins with apparent molecular masses of 100 kDa and 75 kDa respectively . The two proteins are part of a particle termed PM/Scl localized in the granular component of the nucleolus . The predominant immunoreactivity of the PM/Scl sera was shown to be directed against the 100 kDa protein . The cDNA of the 100 kDa protein has been cloned recently and its immunogenic regions have been partially mapped using recombinant proteins . Thus far the localization of antigenic determinants on polypeptides has been done by expressing defined cDNA fragments in bacteria or by synthesizing overlapping short peptides and probing their immunoreactivity with antibodies . Here we present an alternative approach to localize autoimmune epitopes using sera containing polyclonal antibodies and gene-fragment phage display libraries . For epitope fine mapping of the PM/Scl-100 protein random fragments of the corresponding cDNA were cloned into the PIII protein of fUSE-5 . These gene-fragment phage display libraries were incubated with affinity purified anti-PM/Scl-100 antibodies to enrich for epitope-displaying phages . All PM/Scl sera tested recognized 23 consecutive amino acids (229-251) encoded by four overlapping fUSE-5 clones, suggesting that a major epitope is contained within the 23 amino acids . In addition a minor epitope was localized in a region of 21 amino acids (775-795) encoded by two overlapping fUSE-5 clones since only three out of the seventeen sera reacted with this amino acid sequence . Additional fine mapping of the major epitope was done using synthetic oligopeptides . Thus, a stretch of 16 amino acids at position 229-244 could be identified as a major epitope on the deduced PM/Scl-100 amino acid sequence. Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 13398 - 403 Addition of destabilizing poly (A)-rich sequences to endonuclease cleavage sites during the degradation of chloroplast mRNA; Lisitsky I et al.; In this work, we report the posttranscriptional addition of poly(A)-rich sequences to mRNA in chloroplasts of higher plants . Several sites in the coding region and the mature end of spinach chloroplast psbA mRNA, which encodes the D1 protein of photosystem II, are detected as polyadenylylated sites . In eukaryotic cells, the addition of multiple adenosine residues to the 3' end of nuclear RNA plays a key role in generating functional mRNAs and in regulating mRNA degradation . In bacteria, the adenylation of several RNAs greatly accelerates their decay . The poly(A) moiety in the chloroplast, in contrast to that in eukaryotic nuclear encoded and bacterial RNAs, is not a ribohomopolymer of adenosine residues, but clusters of adenosines bounded mostly by guanosines and rarely by cytidines and uridines; it may be as long as several hundred nucleotides . Further analysis of the initial steps of chloroplast psbA mRNA decay revealed specific endonuclease cleavage sites that perfectly matched the sites where poly(A)-rich sequences were added . Our results suggest a mechanism for the degradation of psbA mRNA in which endonucleolytic cleavages are followed by the addition of poly(A)-rich sequences to the upstream cleavage products, which target these RNAs for rapid decay. Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 13212 - 6 Molecular basis for the exquisite sensitivity of Mycobacterium tuberculosis to isoniazid; Zhang Y et al.; The exceptional sensitivity of Mycobacterium tuberculosis to isonicotinic acid hydrazide (INH) lacks satisfactory definition . M . tuberculosis is a natural mutant in oxyR, a central regulator of peroxide stress response . The ahpC gene, which encodes a critical subunit of alkyl hydroperoxide reductase, is one of the targets usually controlled by oxyR in bacteria . Unlike in mycobacterial species less susceptible to INH, the expression of ahpC was below detection limits at the protein level in INH-sensitive M . tuberculosis and Mycobacterium bovis strains . In contrast, AhpC was detected in several series of isogenic INH-resistant (INHr) derivatives . In a demonstration of the critical role of ahpC in sensitivity to INH, insertional inactivation of ahpC on the chromosome of Mycobacterium smegmatis, a species naturally insensitive to INH, dramatically increased its susceptibility to this compound . These findings suggest that AhpC counteracts the action of INH and that the levels of its expression may govern the intrinsic susceptibility of mycobacteria to this front-line antituberculosis drug. Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12817 - 21 A three-hybrid system for detecting small ligand-protein receptor interactions; Licitra EJ et al.; Small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine . We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo . This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined . The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506 . Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone-FK506 heterodimer . The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506 . Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor-LexA DNA binding domain fusion protein in the presence of dexamethasone-FK506 heterodimer . We isolated overlapping clones of human FKBP12 . These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors. Biochemistry, 1996 Nov 12, 35(45), 14203 - 15 Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ; de Pereda JM et al.; The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% alpha helix, 21% beta sheet, and 45% other secondary structure . Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure . Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD {Rost, B., & Sander, C . (1993a) J . Mol . Biol . 232, 584-599} . The predicted secondary structures average of 33% alpha helix, 24% beta sheet, and 43% loop for the alpha beta dimer . The predictions have been compared with sites of limited proteolysis by 12 proteases at the surfaces of the heterodimer and taxol-induced microtubules {de Pereda, J . M., & Andreu, J . M . (1996) Biochemistry 35, 14184-14202} . From 24 experimentally determined nicking sites, 18 are at predicted loops or at the extremes of secondary structure elements . Proteolysis zone A (including acetylable Lys40 and probably Lys60 in alpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extending between residues 167 and 183 in both chains) are accessible in microtubules . Proteolysis zone C, between residues 278 and 295, becomes partially occluded in microtubules . The alpha-tubulin nicking site Arg339-Ser340 is at a loop following a predicted alpha helix in proteolysis zone D . This site is protected in taxol microtubules; however, a new tryptic site appears which is probably located at the N-terminal end of the same helix . Zone D also contains beta-tubulin Cys354, which is accessible in microtubules . Proteolysis zone E includes the C-terminal hypervariable loops (10-20 residues) of each tubulin chain . These follow the two larger predicted helical zones (residues 372-395 and 405-432 in beta-tubulin), which also are the longer conserved part of the alpha- and beta-tubulin sequences . Through combination of this with other biochemical information, a set of surface and distance constraints is proposed for the folding of beta-tubulin . The FtsZ sequences are only 10-18% identical to the tubulin sequences . However, the predicted secondary structures show two clearly similar (85-87 and 51-78%) regions, at tubulin positions 95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300, respectively . The first region is flanked by tubulin proteolysis zones A and B . It consists of a predicted loop1-helix-loop2-sheet-loop3-helix-loop4-sheet fold, which contains the motif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif (SAG)GGTG(SAT)G (loop3) . A simple working model envisages loop1 and loop3 together at the nucleotide binding site, while loops 2 and 4 are at the surface of the protein, in agreement with proteolytic and antigenic accessibility results in tubulin . The model is compatible with studies of tubulin and FtsZ mutants . It is proposed that this region constitutes a common structural and evolutionary nucleus of tubulins and FtsZ which is different from typical GTPases. Biochemistry, 1996 Nov 12, 35(45), 14109 - 17 Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis; Gong J et al.; 5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria . The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor . We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site {Gong, J., & Ferreira, G . C . (1995) Biochemistry 34, 1678-1685} . We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants . Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop . In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template . G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor . While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase . The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site . G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme . Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif. Mutat Res, 1996 Nov 4, 358(2), 171 - 83 Hypersensitivity to very-low single radiation doses: its relationship to the adaptive response and induced radioresistance; Joiner MC et al.; There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents . Phenomenologically, there are two ways in which these induced mechanisms operate . First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose . This has been termed the adaptive response . Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered . This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases . Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo . There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the 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 although exact homology remains to be tested . 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. Z Geburtshilfe Neonatol, 1996 Nov-Dec, 200(6), 221 - 6 {Fetal pulse oximetry sub partu: on morbidity of infection and acceptance in intrapartum monitoring}; Butterwegge M et al.; 156 mothers and their newborns (Group A), whose deliveries were monitored using cardiotocography and fetal pulse oximetry, were investigated during and after delivery regarding amnioninfection as well as changes in morbidity und compared to matched controls (Group B) . The parameters observed were temperature during labor and delivery and after delivery, infection parameters of mother and baby, bacterial smears of the vagina before placement of the oximetry sensor and smears of the sensor tip when the evaluation was concluded . An amnioninfection syndrome was registered twice in group A and three times in control group B . In 22 cases the evaluation of the smears showed an increase of bacterial growth or additional bacteria, but in no case an amnioninfection syndrome was noted . Three of the newborns in Group A who had an infection after delivery showed negative bacterial smears from the sensor tip . The temperatures of the mothers during and after delivery in Group A were not significantly different from Group B . The results show that the intrauterine application of a sensor even for longer periods of time does not result in an increase of maternal or fetal infection morbidity during labor and delivery and after delivery . In addition, 65 patients were evaluated with an anonymous questionnaire after delivery regarding their acceptance of this new method . 88% of the patients were satisfied with the procedure and stated that they felt an additional sense of security through this supplementary method. Equine Vet J, 1996 Nov, 28(6), 480 - 8 The role of computed tomography in evaluation of subchondral osseous lesions in seven horses with chronic synovitis; Hanson JA et al.; Seven horses with severe, persistent lameness of sudden onset were evaluated with scintigraphy and/or computed tomography . The lameness was localised to the front fetlock joint in 2 horses and to the tibiotarsal joint in 5 horses . Five of the horses had a history of intra-articular injections of the involved joint prior to presentation . All horses had effusion of the affected joint and were positive to flexion tests . Intraarticular anaesthesia eliminated or improved the lameness in 4 cases and a nerve conduction block proximal to the affected joint improved the lameness in another . Cytology examination of fluid from affected joints identified normal joint fluid (one horse) or elevations in nucleated cell counts of 0.9 x 10(9)/l-36.8 x 10(9)/l and total protein 20-42 g/l (6 horses) . The joint fluid of 2 of these horses cultured positive for bacteria . Initial radiographs were either normal (4 cases) or the changes seen were not sufficient to explain the degree of lameness . In the 6 cases where scintigraphy was performed, intense focal isotope uptake was found in the suspected region, which corresponded to the proximal portion of the first phalanx (2 cases), distal tibia (2 cases), or talus (3 cases) . Computed tomography (CT) was performed because occult fracture or osteomyelitis was suspected; and knowledge of the precise anatomical location of the lesion was considered necessary to assess the need for surgery and to plan the surgical approach . Hypodense focal lesions with hyperdense haloes were found in the subchondral bone deep to the sagittal groove of the first phalanx (P1) (2 cases) in the cochlea of the distal tibia (2 cases), and in the intertrochlear portion of the talus (3 cases) . Communication between the lesion and the joint space was demonstrated by CT in 5 cases . Post mortem examination of one case revealed synovitis and a chronic bone abscess (Brodie's abscess) communicating with the joint space. Pept Res, 1996 Nov-Dec, 9(6), 262 - 8 Antifungal activity of synthetic 15-mer peptides based on the Rs-AFP2 (Raphanus sativus antifungal protein 2) sequence; De Samblanx GW et al.; Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth . A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized . Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL) . The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium . When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold . By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands . A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity . Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis. Semin Liver Dis, 1996 Nov, 16(4), 343 - 8 Classification, etiology, and considerations of outcome in acute liver failure; Williams R; Clinical descriptions of fulminant hepatic failure as originally reported, along with the subgroups of subfulminant and late onset hepatic failure identified later, are considered in relation to the proposed new classification of hyperacute, acute, and subacute liver failure . This reflects different clinical patterns of illness, etiology, and most importantly, prognosis . In addition to the defining state of encephalopathy and other manifestations directly related to the severe derangement in function and structure of the liver, the constellation of clinical symptoms and signs in acute liver failure (ALF) includes, to varying degrees, those of multiorgan failure . The latter develops because of tissue hypoxia from microcirculatory changes consequent on endotoxemia, and activation of macrophages and release of cytokines as a result of secondary bacteria infection due to an early failure of host defenses to infection in ALF . Paracetamol overdose-the commonest cause of acute liver failure in the United Kingdom-is increasing in frequency in other Western countries, but fulminant viral hepatitis is the most frequent etiology worldwide . Marked geographical variations are seen in the frequency with which the viral types A to E are implicated . Whereas hepatitis C is the major cause of ALF in Japan and the Far East, fulminant hepatitis C is seen rarely in America and European countries where most series show that in about one third of cases of presumed viral ALF, no specific agent can be identified . Over the past 10 years, the survival of those with grade 3 to 4 encephalopathy has shown a steady rise as a result of improvements in medical care, quite apart from the introduction and now widespread availability of transplantation for the treatment of this condition . As shown by a number of groups, a variety of different hematologic, biochemical, and clinical features can be used as predictive indices of the likely outcome and in determining the approach to treatment. Gut, 1996 Nov, 39(5), 639 - 48 Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis; Sakagami T et al.; BACKGROUND/AIMS: The role of host factors has been neglected in studies of the pathogenesis of Helicobacter associated disease . The aim of this study was to assess the response of different mouse strains to infection with a single strain of Helicobacter felis . METHOD: Six strains of inbred mice were infected with the identical H felis culture and were killed at one month, two months, and six months after infection to assess histopathological changes . In addition, two strains of mice were infected with a mouse adapted strain of H pylori and examined at six months after infection . RESULTS: In SJL, C3H/He, DBA/2, and C57BL/6 infected mice, severe to moderate chronic active gastritis was observed only in the body of the stomach, which increased in severity over time with specialised cells in the body glands being replaced . As the severity of this damage in the body increased and atrophic changes were seen, the level of bacterial colonisation of the antrum decreased . In contrast, in BALB/c and CBA mice, there was only mild gastritis in the antrum, no remarkable changes were detected in their body mucosa, and no atrophy was seen over time . In both these strains of mice, heavy bacterial colonisation was seen, which tended to increase over the period of the experiment . Of particular importance in this experiment was that bacterial colonisation was mainly restricted to the antrum yet the atrophy, when present, was only observed in the body of the stomach . H pylori infected C3H/He mice showed moderate colonisation of the antrum, which persisted up to six months with little development of atrophy . In contrast, H pylori in C57BL/6 mice showed excellent colonisation of the antrum at two months but six months after infection there was moderate to severe body atrophy, which was associated with a loss of bacteria from the antrum . CONCLUSIONS: These findings challenge current concepts of the development of Helicobacter induced atrophy in that active chronic gastritis of antrum or the body mucosa, or both, is not a prerequisite . They also suggest an autoimmune basis for the pathology although no autoantibody or antibody to the H+/K+ ATPase was detected . Loss of infecting helicobacters from the stomach together with development of an atrophic gastritis in the body of the stomach is similar to the pattern found in certain H pylori infected human subjects. Radiats Biol Radioecol, 1996 Nov-Dec, 36(6), 874 - 82 {A single mechanism for the initiation of different effects of low doses of ionizing radiation}; Eidus LKh; Main regularities of different endpoints of low dose effects (adaptive response, stimulation of proliferation, special radiosensitivity of lymphoid cells, and others) have been examined . It has been shown that these endpoints have a commonness for the dose interval, the shape of the dose-effect curve, the reverse effect of dose rate, non-specificity toward initiating agents, and others . An explanation is suggested for the common mechanism of the initiation of all the studied low dose effects, basing on the theory of the non-specific reaction of cell to external influences . It is concluded that initiation of the low dose effects is conditioned by radiation induced damage of functions of plasmic and internal membranes. J Toxicol Sci, 1996 Nov, 21 Suppl 3, 495 - 504 {Oral single-dose toxicity study of a new antineoplastic agent S-1, and its components, CDHP, and Oxo}; Hayashi T et al.; S-1, an antineoplastic formulation of a fluorinated pyrimidine derivative containing tegafur (FT), CDHP, and potassium oxonate (Oxo) in a molar ratio of 1:0.4:1, was recently developed by Taiho Pharmaceutical Co., Ltd., with the aim of prolonging the effective plasma concentration of 5-fluorouracil (5-FU) over that produced by FT alone and reducing its dose-limiting gastrointestinal toxicity . As a part of the S-1 toxicity study, the single-dose toxicity of S-1 as well as that of its components, CDHP and Oxo, was investigated in mice, rats, and dogs . The following results were obtained . 1 . In mice and rats, excretion of diarrheal stools, salivation, and alopecia were observed after S-1 administration . In severe cases, the animals subsequently showed emaciation due to weight loss or suppressed weight gain, decreased spontaneous motor activity, an anemic appearance, bradypnea, prone position, and death . In the CDHP and Oxo treatment groups of rats, the only toxic signs were soft or diarrheal stools on the dosing day . 2 . In dogs, vomiting and excretion of diarrheal, mucous, or soft stools was observed after S-1 administration . In the CDHP and Oxo treatment groups, excretion of soft and diarrheal stools and vomiting were observed relatively frequently from the dosing day until day 1 . 3 . In the pathological examination of the animals given S-1, mice and rats showed pulmonary congestion/edema, dark red discoloration of the mesenteric lymph nodes, atrophy of lymphatic tissues such as the thymus and lymph nodes, decreases of lymphocytes in the splenic white pulp and mesenteric lymph nodes, a decrease in bone marrow cells, congestion of the glandular stomach, and aggregates of bacteria in the lung, liver, or spleen . In dogs, abnormal changes were observed mainly in the lymphatic organs such as the thymus and lymph nodes . 4 . The LD50 values of S-1 in terms of the amount FT they contained were estimated to be 549 mg/kg for mice(male), 441-551 mg/kg for rats (both sexes) and about 53 mg/kg for dogs (male) . The LD50 values of CDHP and Oxo were 2000 mg/kg or higher for both rats (both sexes) and dogs (male) . 5 . Hematopoietic and lymphatic impairments, immunosuppression associated with respiratory were considered to be the cause of death from S-1 . The toxicity of S-1 reflects the toxicity of 5-FU and was not found the different toxicity by the addition of CDHP and Oxo. Plasmid, 1996 Nov, 36(3), 169 - 81 Wild-type strains of Dictyostelium discoideum can be transformed using a novel selection cassette driven by the promoter of the ribosomal V18 gene; Wetterauer B et al.; Almost all methods for transformation of the social ameba Dictyostelium discoideum rely on axenic growth, that is, growth in a synthetic medium, for at least part of the procedure . Axenic growth requires several mutations . Here we describe a procedure that can be used to transform wild-type strains which are able to grow only on the natural food source, bacteria . The method relies on a new selection cassette driven by the V18 promoter, a promoter that we show is substantially more active during growth on bacteria than the actin-6 promoter, which is widely used for axenic transformation . The procedure gives transformation frequencies of about 10(-5) with both strains Ax2 (capable of axenic growth) and NC4 (capable of growth only on bacteria) . Using this vector, we have obtained NC4 strains carrying several beta-galactosidase reporter cassettes . Our vector can also be used in axenic transformations. Vet Immunol Immunopathol, 1996 Nov, 54(1-4), 191 - 5 The respiratory immune system of pigs; Pabst R; Respiratory tract infections with bacteria like Actinobacillus pleuropneumoniae are extremely common in pigs and are of major veterinary relevance . The respiratory tract can be divided into the upper part, consisting of the nose, pharynx, larynx and trachea, and the lower part, consisting of the different parts of the lung . After bronchoscopy and bronchoalveolar lavage (BAL) had been established for pigs, interest grew in the unspecific parts of the immune system of the respiratory tract (such as macrophages, mast cells, the mucociliary function) and the specific immune system, consisting of the different lymphocyte subsets . In contrast to the rodent and human lung, the lung of the pig contains large numbers of intravascular macrophages with a high clearance capacity . The main focus of this paper is the localization, subset composition and quantification of lymphocytes in the pig lung: the intravascular and interstitial pool and the lymphocytes in the bronchial epithelium and lamina propria including bronchus-associated lymphoid tissue form the major compartments . In the BAL only a small proportion of nucleated cells are lymphocytes . The effects of age, antigen exposition, immunization and infection on the lymphocyte distribution in the pig lung are presented . In addition to veterinary aspects, the lung of pigs can also serve as a model for diseases in humans. Lett Appl Microbiol, 1996 Nov, 23(5), 350 - 4 Specific primers for detection of Alicyclobacillus acidoterrestris by RT-PCR; Yamazaki K et al.; The reverse transcription-polymerase chain reaction (RT-PCR), after a short enrichment culture, was used to detect Alicyclobacillus acidoterrestris in acidic beverages . Two specific primers were selected from the region of V2 and V4 on 16S rRNA gene . With this primer set, 294-bp fragments from A . acidoterrestris could be amplified . The detection limit of the RT-PCR with the FHLP filters was about 10-1 fg of pure total RNA per reaction . Juice samples inoculated with 10(4) cfu of A . acidoterrestris per ml were RT-PCR positive without enrichment . However, after 15 h of enrichment, the samples inoculated with 2-3 cfu ml-1 were positive . This RT-PCR culture assay would enable rapid and specific detection of strains of A . acidoterrestris in acidic beverages. Plant Mol Biol, 1996 Nov, 32(4), 685 - 92 A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny; Vogel H et al.; Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen . We have analyzed one of the genes essential for this process, secY . A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid . Unlike the situation seen in bacteria the C . caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons . A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet . In this analysis plastid secY genes fall into two classes . One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart . Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga . All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues . We postulate that the two classes of secY genes are paralogous, i.e . their gene products are involved in different protein translocation processes . Based on this assumption a model for the evolution of the plastid sec apparatus is presented. Nutrition, 1996 Nov-Dec, 12(11-12 Suppl), S78 - 81 Glutamine: an essential amino acid for the gut; van der Hulst RR et al.; Glutamine is a non-essential amino acid which is produced in sufficient amount by the healthy human body . From experimental work it is known that glutamine is an important nutrient for rapidly dividing cells such as cells from the immune system and the gut . During several conditions a lack of glutamine may occur . This will result in functional disturbances of the immune system and/or the gut . Glutamine is produced mainly by the muscle tissue . A decrease in muscle mass during nutritional depletion may result in decreased glutamine production capacity . Furthermore during critical illness, there is an increased demand for glutamine probably as a result of an increased utilization by the immune system . In addition, patients receiving standard parenteral nutrition do not receive glutamine, until recently, commercial parenteral nutrition did not contain glutamine because of instability of this amino acid during prolonged storage . One of the important functions of the gut is to prevent migration of bacteria and/or toxins from the gut lumen into the systemic circulation . A lack of glutamine may result in deterioration of this intestinal barrier . Supplementation of glutamine to certain patients could be essential . The relation between glutamine and the gut in several situations (nutritional depletion, critical illness, parenteral nutrition) is discussed in this paper. Carcinogenesis, 1996 Nov, 17(11), 2477 - 86 Histogenesis and the role of p53 and K-ras mutations in hepatocarcinogenesis by glyceryl trinitrate (nitroglycerin) in male F344 rats; Tamano S et al.; Glyceryl trinitrate (GTN) was previously reported to induce hepatocellular carcinoma (HCC) in rats after prolonged feeding . The present experiments were undertaken to evaluate the histogenesis and molecular biology of these tumors and the possible role of nitric oxide (NO), a GTN metabolite, in their development . Male F344 rats received a single i.g . intubation of GTN (1.2 g/kg) at 6 weeks of age and/or a diet containing 1% GTN from 8 weeks of age until necropsy, i.e . for up to 78 weeks . Some animals were subjected to 2/3 partial hepatectomy (PH) at 9 weeks of age . Five sequential sacrifices (14, 32, 52, 78 and 84 weeks of age) were performed . No liver tumors developed in control rats or in rats that received GTN only by a single i.g . intubation, even when intubation was followed by PH . Preneoplastic foci, mainly of clear cell and mixed cell type (identified as positive for glutathione S-transferase placental form) were found from 14 weeks of age in rats receiving GTN in the diet . Focal eosinophilic areas (atypical foci) composed of atypical hepatocytes that often extended into the veins were observed beginning at 52 weeks of age . Some mixed hepatocholangiocellular adenomas and carcinomas arose in eosinophilic lesions . HCCs were seen beginning at 78 weeks of age, but only in rats receiving dietary GTN . Incidence of HCC in the latter animals was 50-75% . Most HCCs were well differentiated . The carcinogenic effect of GTN given in the diet was not affected by prior intubation of a large single dose followed by PH . No p53 mutations were found in 18 tumors but K-ras point mutations, all within codon 12, were found in 8/18 tumors, mostly those with cholangiocellular elements . These were first or second position G-->T transversions or second position G-->A transitions . While these mutation types have also been commonly seen in bacteria after NO-related DNA damage, the fact that tumors arose only on prolonged feeding of this potently bioactive agent at massive doses seems consistent with a more complex mechanism involving multiple (i.e . genetic and/or epigenetic) factors in carcinogenesis by GTN. J Clin Periodontol, 1996 Nov, 23(11), 982 - 8 Risk indicators for periodontal disease in a racially diverse urban population; Alpagot T et al.; A cross-sectional study of 117 subjects from a dental clinic serving a diverse population (i.e., Whites, African-Americans, Native-Americans, and Asians) was performed to evaluate risk indicators of periodontal disease . Gingival crevicular fluid (GCF) and subgingival plaque were taken at the same visit from 4 posterior sites of the most diseased sextant in each subject . Age, smoking packyears, beta-glucuronidase (beta G), neutrophil elastase (NE), myeloperoxidase (MPO), Fusobacterium nucleatum (F . nucleatum), and Porphyromonas gingivalis (P . gingivalis) were significantly (p < 0.05-0.005) correlated with attachment loss . Probing depth was significantly correlated with smoking packyears, beta G, NE, MPO, F . nucleatum and Prevotella intermedia (P . intermedia) (p < 0.05-0.005) . Mean NE value of Whites was lower than the mean NE values of African-Americans, Native-Americans and Asians (p < 0.05) . Whites had a lower mean beta G value compared to African Americans, and a lower mean MPO value compared to African Americans and Native Americans . The %s of patients positive for F . nucleatum, P . intermedia and Eikenella corrodens (E . corrodens) were higher in Native Americans compared to Whites . Step-wise multiple regression analysis was performed to construct models for the estimation of probing depth and attachment loss . The most parsimonious regression models which had the best R2 values included the following variables and accounted for the indicated % of variability: models 1 and 2: beta G, race, and F . nucleatum accounted for 50% of the variability in mean probing depth and 39% of the variability in a single site (first molar) for probing depth, respectively; model 3: age, beta G, and F . nucleatum accounted for 53% of the variability in mean attachment loss; model 4: age, NE, and F . nucleatum explained 35% of the variability in a single site (first molar) for attachment loss . The results suggest that age, race, smoking packyears, beta G, NE, MPO, F . nucleatum, P . gingivalis and P . intermedia are risk indicators for periodontal disease in this racially diverse urban population . Regression models which include multiple variables (i.e., demographic factors, GCF enzymes and periodontopathic bacteria) can be used to estimate periodontal disease status. Trends Microbiol, 1996 Nov, 4(11), 453 - 5 How do macrophages distinguish the living from the dead? Cheers C, Zhan Y. Injection of live but not dead bacteria induces a wave of IL-12 and subsequently, IFN-gamma production . Surprisingly, in vitro, both live and dead bacteria elicit IL-12 from macrophages . Better understanding of how macrophages distinguish live from dead bacteria would help explain this difference and possibly bypass the need for live vaccines against intracellular bacteria. Scand J Immunol, 1996 Nov, 44(5), 535 - 9 Natural polyreactive IgA and IgM autoantibodies in human colostrum; Vassilev TL et al.; Secretory antibodies against bacteria and viruses in human colostrum and milk are known to be important protective factors for the breast-fed infant . The authors have shown by enzyme immunoassay that colostrum contains IgA and IgM antibodies to a number of autoantigens: native DNA, actin, myosin, myoglobin, laminin, transferrin and thyroglobulin . These antibodies were polyspecific-those with anti-DNA reactivity immunopurified on a DNA-cellulose affinity column bound to a panel of self- and environmental antigens . The levels of natural autoantibodies in the immunoglobulin fraction of human colostrum were 3-10 times lower (when presented as antibody activity per microgram of immunoglobulin) than in the immunoglobulin fraction of serum . The biological significance of the presence of B cells with autoantibody specificity in the mammary gland and of natural autoantibodies in colostrum and milk is not clear . It has been suggested that self-reacting autoantibodies in serum play a major role in the selection of the pre-immune B-cell repertoire and in the maintenance of the immune homeostasis . The authors hypothesize that the natural autoantibodies in colostrum and milk may contribute to the selection process of physiological repertoire during the early postnatal period in breast-fed infants . This could explain the lower frequency of allergic, inflammatory and autoimmune diseases and lymphomas which is seen in their later life when compared with that observed in children who have been formula-fed after birth. Eur J Biochem, 1996 Nov 1, 241(3), 716 - 22 Structure and biological activity of chemically modified nisin A species; Rollema HS et al.; Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb) . Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry . Five constituents, {2-hydroxy-Ala5}nisin, {Ile4-amide,pyruvyl-Leu6}des-Dha5-nisin, {Met(O)21}nisin, {Ser33}nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample . A further species, {2-hydroxy-Ala5}nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution . These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues . The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives . The growth-inhibiting activity of the modified nisins towards different bacteria was determined . The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin . {Met(O)21}nisin and {Ser33}nisin show moderate or no reduction in biological activity. Curr Biol, 1996 Nov 1, 6(11), 1377 - 80 Genomes: Methanococcus jannaschii and the golden fleece; Garrett RA; The total genome sequence of the archaeon Methanococcus jannaschii completes the trilogy of genomes for the three domains of life-Archaea, Bacteria and Eucarya . It will have far-reaching consequences for evolutionary studies and for the way in which experimental biology is performed. Mol Microbiol, 1996 Nov, 22(3), 473 - 80 Transcriptional regulation of zwf, encoding glucose-6-phosphate dehydrogenase, from the cyanobacterium Nostoc punctiforme strain ATCC 29133; Summers ML et al.; The gene encoding glucose-6-phosphate dehydrogenase (G6PD), zwf, in Nostoc punctiforme strain ATCC 29133 is part of a four-gene operon that also encodes fructose bisphosphatase (fbp), transaldolase (tal) and a gene product termed OpcA, which is contranscribed with zwf and essential for G6PD activity . The effect of exogenous nitrogen and carbon sources on transcription of these genes was investigated . Growth in the presence of ammonium yielded low levels of transcripts encoding all genes of the operon, while growth under nitrogen-fixing conditions resulted in a large increase of transcripts encoding for fbp and zwf-opcA . When cells are grown in the presence of fructose, levels of transcripts encoding tal and zwf-opcA were increased, relative to levels in ammonium-grown cells . These results indicate that this facultatively heterotrophic cyanobacterium can respond to changes in its environment by altering transcription of genes involved in carbon catabolism . Primer extension identified five 5' ends corresponding to the major regulated transcripts which we conclude arise from independent transcriptional start points. Mol Microbiol, 1996 Nov, 22(3), 393 - 404 A common export pathway for proteins binding complex redox cofactors? Berks BC. The precursor polypeptides of periplasmic proteins binding seven types of redox cofactor have unusually long signal sequences bearing a consensus (S/T)-R-R-x-F-L-K motif immediately before the hydrophobic region . Such "double-arginine' signal sequences are not, in general, found on the precursors of other periplasmic proteins . It is suggested that precursor proteins with double-arginine signal sequences share a common specialization in their export pathway . The nature of this specialization, the structure of the double-arginine signal sequences, and the possible relationship with the double-arginine signal peptide-dependent thylakoid import pathway are discussed. Res Vet Sci, 1996 Nov, 61(3), 183 - 6 Bovine leucocyte adhesion deficiency: a review of a modern disease and its implications; Gerardi AS; Bovine leucocyte adhesion deficiency (BLAD) is a genetic disease of cattle which affects the hematopoietic system . In the last decade BLAD has become a disease of economic importance in the dairy industry . This review describes the chronological developments and thinking that led to the elucidation of BLAD as a disease distinct from previous models in canine and human populations . All species affected show signs of chronic and recurrent infections . Necrotic and/or gangrenous infections of soft tissues are prevalent, in addition to secondary infections with bacteria or fungi . Low birthweight and unthriftiness are key signs in all species affected by leucocyte adhesion deficiency (LAD) . Dermatomycoses and impaired pus formation are also common findings . The physiological basis for BLAD is a deficiency in the chemotactic and phagocytic properties of leucocytes and particularly neutrophils . The inhibition of diapedesis in the inflammatory response prevents normal immune reactions to invading pathogens . Chronic infections are a consequence of the faulty immune mechanisms . The biochemical aetiology of BLAD involves cell surface glycoprotein molecules known as integrins . These are responsible for the cell-cell interactions necessary for neutrophils to adhere to vascular endothelium in a normal individual . Experiments with monoclonal antibodies to block LFA-1, Mac-1, and p150,95 (three integrins vital for cell-cell interactions) mimic BLAD symptomatology and have led to the discovery of the reciprocal intercellular adhesion molecule (ICAM) . Through pedigree analysis and biochemical detection with restrictive endonucleases, BLAD has been isolated genetically to a single gene locus . The economic significance and prophylaxis of the disease are briefly discussed . In addition, the beneficial aspects of the study of BLAD are considered . There are advantages in producing a BLAD-like state for preventing transplant rejection, ischaemia-reperfusion injury and other problems arising from the deleterious effects of the inflammatory response. Plant Physiol, 1996 Nov, 112(3), 905 - 17 De novo purine synthesis in Arabidopsis thaliana . II . The PUR7 gene encoding 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase is expressed in rapidly dividing tissues; Senecoff JF et al.; The small genome size and excellent genetics of Arabidopsis, as well as the ease with which it is transformed, make it a superb candidate for molecular genetic studies of the purine biosynthetic pathway . Herein we report the isolation, physical characterization, and dissection of the expression patterns of the single gene encoding 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole synthetase . This enzyme, encoded by the PUR7 gene, catalyzes aspartate addition at the alpha-amino group to the growing purine backbone . The expression of the PUR7 as directed by the 5' region, containing the promoter, mRNA leader, and leader intron, was examined in Arabidopsis using a transgenic reporter system . Our analysis demonstrates that the highest level of purine biosynthesis occurs in mitotically active tissues of the plant . Furthermore, purine biosynthesis appears to be under developmental and hormonal regulation . Inhibition of purine biosynthesis using substrate analogs results in arrested plant development and induction of purine gene expression . Purine nucleotides and their derivatives provide multiple cofactors for a variety of metabolic processes . Our findings begin to identify some of the regulatory mechanisms that affect the production of purine nucleotides in Arabidopsis and may give important insights into nitrogen metabolism in general. Neuron, 1996 Nov, 17(5), 911 - 20 Regulation of nuclear entry of the Drosophila clock proteins period and timeless; Saez L et al.; Two genes, period (per) and timeless (tim), are essential for circadian rhythmicity in Drosophila . The encoded proteins (PER and TIM) physically interact . Here, it is shown that TIM and PER accumulate in the cytoplasm when independently expressed in cultured (S2) Drosophila cells . However, the proteins move to the nuclei of these cells if coexpressed . Domains of PER and TIM have been identified that block nuclear localization of the monomeric proteins . In vitro protein interaction studies indicate that the sequence inhibiting the nuclear accumulation of PER forms a binding site for TIM . The results indicate a mechanism for controlled nuclear localization in which suppression of cytoplasmic localization is accomplished by direct interaction of PER and TIM . No other clock functions are required for nuclear localization . The findings suggest that a checkpoint in the circadian cycle is established by requiring cytoplasmic assembly of a PER/TIM complex as a condition for nuclear transport of either protein. J Dairy Res, 1996 Nov, 63(4), 517 - 24 Influence of pulsationless milking on teat canal keratin growth and turnover; Lacy-Hulbert SJ et al.; In two separate experiments, the effects of pulsationless milking and milking vacuum on the rate of keratin removal from the teat canal were determined . Sixteen cows were milked with or without pulsation for either a single milking or for eight milkings . Milking without pulsation removed 10% of keratin present before milking, significantly less than milking with pulsation, which removed 32% . After eight milkings (4 d) without pulsation, up to 20% more keratin had accumulated within the teat canal but the rate of keratin regeneration reduced significantly upon return to pulsation milking . In a second experiment, ten cows were milked at 45 or 55 kPa and without pulsation . Only the absence of pulsation significantly reduced keratin loss during milking . Keratin loss during milking appears to be controlled by liner compression rather than by the rate of milk flowing through the teat canal . Pulsationless milking may increase penetrability of the teat canal to bacteria by reducing the natural rate of keratin removal during milking and reducing the rate of keratin regeneration. J Bacteriol, 1996 Nov, 178(22), 6647 - 9 Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus; Udo H et al.; Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus . The effect of Pkn2 overexpression on development of M . xanthus was examined by expressing pkn2 under the control of a kanamycin promoter . Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain . Overexpressed Pkn2 was located almost exclusively in the membrane fraction, suggesting that Pkn2 is a transmembrane receptor-type protein Ser/Thr kinase . The PKm/pkn2 strain formed fruiting bodies more slowly than the wild-type strain, in contrast to a Pkn2 deletion strain, the delta pkn2 strain, which developed faster than the wild-type strain . However, spore production was reduced in both the PKm/pkn2 and delta pkn2 strains . These data suggest that Pkn2 functions as a negative regulator for fruiting-body formation and that the proper level of Pkn2 is necessary for maximum myxospore yield. J Bacteriol, 1996 Nov, 178(22), 6644 - 6 A novel type of pyridine nucleotide-disulfide oxidoreductase is essential for NAD+- and NADPH-dependent degradation of epoxyalkanes by Xanthobacter strain Py2; Swaving J et al.; Epoxide degradation in cell extracts of Xanthobacter strain Py2 has been reported to be dependent on NAD+ and dithiols . This multicomponent system has now been fractionated . A key protein encoded by a DNA fragment complementing a Xanthobacter strain Py2 mutant unable to degrade epoxides was purified and analyzed . This NADP-dependent protein, a novel type of pyridine nucleotide-disulfide oxidoreductase, is essential for epoxide degradation . NADPH, acting as the physiological cofactor, replaced the dithiols in epoxide conversion. J Bacteriol, 1996 Nov, 178(22), 6587 - 98 GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp; Hill DR et al.; The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity . In N . commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 micromol of O2 mg of chlorophyll a(-1) h(-1) . Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP+ oxidoreductase (PetH or FNR) . A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN . glbN was present in five Nostoc strains in a single copy . Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp . strain PCC 7120, provided no hybridization signals with a glbN probe . A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii . The nifU-nifH intergenic region of Nostoc sp . strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N . commune UTEX 584 . Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired . Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N . commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN . This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp . strain PCC 7942 . In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts . Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase. J Bacteriol, 1996 Nov, 178(22), 6539 - 45 Structural analysis of the Leptospiraceae and Borrelia burgdorferi by high-voltage electron microscopy; Goldstein SF et al.; Spirochetes are an evolutionary and structurally unique group of bacteria . Outermost is a membrane sheath (OS), and within this sheath are the protoplasmic cell cylinder (PC) and periplasmic flagella (PFs) . The PFs are attached at each end of the PC and, depending on the species, may or may not overlap in the center of the cell . The precise location of the PFs within the spirochetal cells is unknown . The PFs could lie along the cell axis . Alternatively, the PFs could wrap around the PC in either a right- or a left-handed sense . To understand the factors that cause the PFs to influence cell shape and allow the cells to swim, we determined the precise location of the PFs in the Leptospiraceae (Leptonema illini) and Borrelia burgdorferi . Our approach was to use high-voltage electron microscopy and analyze the three-dimensional images obtained from thick sections of embedded cells . We found that a single PF in L . illini is located in a central channel 29 nm in diameter running along the helix axis of the right-handed PC . The presence of the PFs is associated with the end being hook shaped . The results obtained agree with the current model of Leptospiraceae motility . In B . burgdorferi, which forms a flattened wave, the relationship between the PFs and the PC is more complicated . A multistrand ridge 67 nm in diameter, which was shown to be composed of PFs by cross-sectional and mutant analysis, was found to extend along the entire length of the cell . We found that the PFs wrapped around the PC in a right-handed sense . However, the PFs formed a left-handed helix in space . The wavelength of the cell body and the helix pitch of the PFs were found to be identical (2.83 microm) . The results obtained were used to propose a model of B . burgdorferi motility whereby backward-propagating waves, which gyrate counterclockwise as viewed from the back of the cell, are generated by the counterclockwise rotation of the internal PFs . Concomitant with this motion, the cell is believed to rotate clockwise about the body axis as shown for the Leptospiraceae. J Bacteriol, 1996 Nov, 178(22), 6427 - 34 The TACAN4TGCA motif upstream from the -35 region in the sigma70-sigmaS-dependent Pm promoter of the TOL plasmid is the minimum DNA segment required for transcription stimulation by XylS regulators; Gallegos MT et al.; Transcription from the TOL plasmid meta-cleavage pathway operon promoter Pm is dependent on the XylS regulator activated by benzoate effectors or after XylS overproduction . We have generated 5' deletions in Pm and have analyzed expression from wild-type and mutant promoters with the wild-type XylS regulator and XylS mutant regulators that stimulated transcription constitutively . We have found that the motifs T(C or A)CAN4TGCA located between -46 and -57 and -67 and -78 with respect to the main transcription initiation point are required for maximal stimulation of transcription from Pm with effector-activated wild-type XylS . Deletion of the farthest TCCA submotif decreased but did not abolish transcription mediated by the pair XylS with 3-methylbenzoate; however, removal of the motif between -67 and -78 resulted in the loss of stimulation by the wild-type regulator . XylSG44S and XylSS229I stimulated high levels of transcription in the absence of effectors from the wild-type promoter and from a mutant promoter exhibiting only the -46 to -57 motif only when an effector was present . The point mutation Pm5U (with C-47 replaced by G {C-47-->G}) and Pm4 (C-68-->G), located in each 3' TGCA submotif of each motif, resulted in a 90% decrease in transcription stimulation with wild-type XylS; however, the mutant XylSS229I stimulated high levels of transcription from the point mutation promoters both in the presence and in the absence of effectors, while mutant XylSG44S suppressed the two point mutations only with 3-methylbenzoate . Overexpression of XylS and XylSG44S allowed the two regulators to stimulate high levels of transcription from the wild-type promoter, the point mutation Pm4 and Pm5U promoters, and deltaPm promoters exhibiting at least the -46 to -57 motif . Therefore the TACAN4TGCA motif between -46 and -57 represents the minimal DNA segment required for stimulation of transcription from Pm. Am Ind Hyg Assoc J, 1996 Nov, 57(11), 1002 - 12 The medical and epidemiologic effects on workers of the levels of airborne Thermoactinomyces Spp . spores present in Australian raw sugar mills; Dawson MW et al.; The objectives of the study were to determine whether there was a significant risk of members of the work force of raw sugar mills developing bagassosis . Airborne Thermoactinomyces sacchari spores were measured to determine whether they were sufficient to cause acute bagassosis, and whether there was any evidence of previous exposure to sufficient airborne T . sacchari spores to cause the development of chronic bagassosis in any of the work force . Monitoring of total airborne bacteria spore concentrations was undertaken in and around two cane sugar mills before, during, and after the 1992 cane processing season . Viable airborne bacteria counts were also obtained to confirm the presence of Thermoactinomyces sacchari . Area or zone samples at various sites around the mills and personal breathing zone samples from selected workers were obtained . The results showed that the total airborne bacteria spore count was lower than similar counts reported in other industries, such as cotton milling and wood chip handling, during normal operations . It was also found that the airborne counts during specific activities that generated higher than usual airborne spore levels were lower than expected from literature reports of handling similar material . Complementary medical examination of the entire full-time work forces of the two mills was carried out on a number of occasions during 1992 . The medical data showed that no cases of acute bagassosis were detected, and that there was no evidence of the development of chronic bagassosis in any members of the work forces of either mill . Therefore, there is no significant risk of workers in the Australian sugar industry developing bagassosis. Trends Neurosci, 1996 Nov, 19(11), 465 - 8 Friedreich's ataxia protein: phylogenetic evidence for mitochondrial dysfunction; Gibson TJ et al.; Friedreich's ataxia is the most common inherited spinocerebellar ataxia . A decade of linkage and physical mapping studies have culminated in the identification of the Friedreich's ataxia gene . The presence of homologues in purple bacterial genomes, but not in other bacteria, allows us to infer a mitochondrial location for frataxin (Friedreich's ataxia protein) on the basis of bacterial phylogeny . Frataxin possesses a non-globular N-terminus domain providing a candidate mitochondrial targeting peptide . Clues to the function of frataxin are provided by the mitochondrial location, a clinically similar ataxia with vitamin E deficiency, and certain neuropathies with mitochondrial DNA instability caused by mutations in nuclear genes. Curr Genet, 1996 Nov, 30(5), 455 - 60 Loss of RNA editing of rps1 sequences in Oenothera mitochondria; Mundel C et al.; We have analysed a region downstream from the atp9 gene in Oenothera mitochondrial DNA which contains an open reading frame of 224 codons . This open reading frame, designated orf224, is co-transcribed with the atp9 gene . In wheat mitochondria, a homologous reading frame (orf174) has been described which is considerably shorter at the N-terminus compared to the putative Oenothera gene . The deduced polypeptides from both species show high similarity to the N-terminal third of ribosomal protein S1 of bacteria . Transcripts of orf174 are edited in wheat mitochondria whereas the similarly conserved cytidine positions in orf224 mRNAs of Oenothera are silent . On the other hand, atp9 sequences, which are located upstream on the same co-transcript, are fully edited . Because of this, we conclude that editing sites are selected independently for mRNA sections . Our results suggest that orf224 represents a transcribed pseudogene in Oenothera . The active gene for a functional ribosomal protein S1 for mitochondria is therefore expected to be present in the nucleus . A nuclear localization for this gene is likewise suggested for Arabidopsis due to the fact that rps1 sequences are absent from the mitochondrial genome. Eur J Immunol, 1996 Nov, 26(11), 2790 - 9 Major histocompatibility complex class I presentation of ovalbumin peptide 257-264 from exogenous sources: protein context influences the degree of TAP-independent presentation; Wick MJ et al.; Peritoneal macrophages from C57BL/6 mice process antigens from bacteria or coated on polystyrene beads for presentation by major histocompatibility complex (MHC) class I molecules . To investigate this antigen processing pathway, peritoneal macrophages from homozygous TAP1-/- mice, which lack the transporter associated with antigen processing (TAP) and are defective in presenting endogenous antigens on MHC class I, were used . TAP1-/- or C57BL/6 macrophages were co-incubated with either bacteria or polystyrene beads containing the 257-264 epitope from ovalbumin {OVA(257-264)}, which binds the mouse class I molecule Kb . The source of the OVA(257-264) epitope was either the Crl-OVA(257-264) (Crl-OVA) fusion protein, the maltose binding protein (MBP)-Crl-OVA fusion protein, native OVA or bacterial recombinant OVA (rOVA); Crl-OVA, MBP-Crl-OVA and rOVA were each expressed in bacteria, and Crl-OVA and MBP-Crl-OVA purified from bacterial lysates and native egg OVA were coated onto polystyrene beads . The data reveal that peritoneal macrophages from C57BL/6 and TAP1-/- mice can process bacteria expressing Crl-OVA, MBP-Crl-OVA and rOVA as well as beads coated with native OVA, purified Crl-OVA, and purified MBP-Crl-OVA and present OVA(257-264) for recognition by OVA(257-264)/Kb-specific T hybridoma cells, albeit with different relative processing efficiencies . The processing efficiency of TAP1-/- macrophages co-incubated with bacteria or beads containing Crl-OVA or MBP-Crl-OVA was reduced approximately three to five times compared to C57BL/6 macrophages, but OVA(257-264) was presented 100 times less efficiently when the source of OVA(257-264) was full-length OVA . Chloroquine inhibition studies showed a differential requirement for acidic compartments in C57BL/6 versus TAP1-/- macrophages, which also depended upon the source of the OVA (257-264) epitope (Crl-OVA versus full-length OVA) . These data suggest that TAP1-/- and C57BL/6 macrophages may process Crl-OVA and full-length OVA in different cellular compartments and that the protein context of the OVA(257-264) epitope influences the extent of TAP-independent processing for MHC class I presentation. Biochem J, 1996 Nov 1, 319 ( Pt 3), 977 - 83 Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24; Ko JH et al.; An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography . The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5% . The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa . The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases . The enzyme was most active at pH 6.0 . The activity was maximal at 73-78 degrees C and its half-life was 30 min at 95 degrees C . Kinetic and regulatory properties were characterized . It was found that AGPase activity could be stimulated by a number of glycolytic intermediates . Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective . The enzyme was inhibited by phosphate, AMP or ADP . ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator . A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues . The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources . It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related. Biochem J, 1996 Nov 1, 319 ( Pt 3), 839 - 42 Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca(2+)-free enzyme with Ca2+, Sr2+ or Ba2+; Goodwin MG et al.; The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3 . Two Ca2+ ions were irreversibly incorporated per alpha 2 beta 2 tetramer . Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+ . Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations . The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+ . The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process . Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process. Biochem J, 1996 Nov 1, 319 ( Pt 3), 829 - 37 Primary structure of the cytosolic beta-glucosidase of guinea pig liver; Hays WS et al.; The cytosolic beta-glucosidase (EC 3.2.1.21) present in the livers of mammalian species is distinguished by its broad specificity for sugars and its preference for hydrophobic aglycones . We purified the cytosolic beta-glucosidase from guinea pig liver and sequenced 142 amino acid residues contained within 12 trypsin digest fragments . Using degenerate oligonucleotide primers deduced from the peptide sequences, a 622 bp cytosolic beta-glucosidase cDNA was amplified by reverse-transcriptase PCR, using total guinea pig liver RNA as template . The 'rapid amplification of cDNA ends (RACE)' method {Frohman (1993) Methods Enzymol . 218, 340-356} was used to synthesize the remaining segments of the full-length cDNA . The complete cDNA contained 1671 nucleotides with an open reading frame coding for 469 amino acid residues . The amino acid sequence deduced from the cDNA sequence included the amino acid sequences of all 12 trypsin digest fragments derived from the purified enzyme . Amino acid sequence analysis indicates that the guinea pig liver cytosolic beta-glucosidase is a Family 1 beta-glycosidase and that it is most closely related to mammalian lactase-phlorizin hydrolase . These results suggest that the cytosolic beta-glucosidase and lactase-phlorizin hydrolase diverged from a common evolutionary precursor. Clin Exp Immunol, 1996 Nov, 106(2), 404 - 9 Neutrophils in mucosal secretion are functionally active; Ebenfelt A et al.; The leucocytes in the secretion on the tonsillar surface have been regarded as inactivated and dying cells with no essential function in the defence of the underlying mucosa . In recent studies, we showed that in acute tonsillitis there are very few, if any, bacteria in the parenchyma, whereas in the secretion on the tonsillar surface there are huge numbers of bacteria and neutrophils, and furthermore, extensive phagocytosis . The present study was performed to elucidate the functional capacity of the neutrophils in the secretion on the tonsillar surface . Secretion samples were taken from the tonsillar surface of healthy volunteers with an imprint technique which allows transfer of secretion from the mucosa to a glass slide with maintained topographic position of the cells . The capacity of the neutrophils to respond to chemotactic stimuli and to phagocytize and further process labelled yeast particles was studied in this in vitro system . The results show that the neutrophils in the secretion have the same properties as in tissue, and also when having arrived in the secretion they can identify and attack new prey. Clin Exp Immunol, 1996 Nov, 106(2), 396 - 403 Abundance of intraepithelial gamma delta T cells in hypertrophic obstructive but not in chronically infected adenoids; Olofsson K et al.; Using quantitative morphometric analysis of immunohistochemically stained tissue sections we compared hypertrophic obstructive adenoids (HOA, n = 10) from children without middle ear disease with chronically infected adenoids (CIA, n = 10) from children with middle ear disease . gamma delta T cell receptor (TCR)+ cells constituted the dominating T cell population in the surface epithelium of HOA, while alpha beta TCR+ cells were the dominating intraepithelial T cell population in CIA . Intraepithelially CD8+ cells dominated over CD4+ cells in both diseases . Intraepithelially B cells were not detected . The cellular composition of follicles, with B cells dominating followed by activated CD4+ alpha beta TCR+ cells, was the same in both groups . However, the number of follicles in CIA was twice as many as in HOA . In the deeper interfollicular areas granulocytes were more abundant in CIA than in HOA . The latter two findings suggest a more pronounced inflammatory response in the adenoids of patients with middle ear disease . There was no significant difference with regard to pathogenic bacterial strains colonizing the adenoid surface when comparing the two patient groups . These results suggest that in patients with HOA gamma delta TCR+ T cells help to maintain the integrity of the surface epithelium, thereby preserving its protective function . On the basis of our results we speculate that CIA have a malfunctioning defence, thereby facilitating long-standing infections deep in the adenoid . This may be the main reason for development of middle ear disease and an indication for adenoidectomy in patients with CIA. Ann Otol Rhinol Laryngol, 1996 Nov, 105(11), 863 - 7 Role of bronchoalveolar lavage in hospitalized pediatric patients; Yagoda MR et al.; Bronchoalveolar lavage (BAL) has been shown to be a rapid, relatively safe, and relatively noninvasive diagnostic procedure . Theoretically, BAL can be performed on all children hospitalized for pneumonia resistant to oral antibiotics, though practically and economically, this is not feasible . A 1-year retrospective review was conducted to define a cost-effective role for BAL in the management of hospitalized children with resistant pneumonia . The data revealed identification of at least one pathogen in 87% of sputum samples and in 95% of BAL specimens . Sputum samples provided the same information as the more invasive BAL technique in 60% of patients who had both sputum and BAL obtained for culture . Recommendations are made for the use of BAL as a diagnostic tool in the hospitalized child with resistant pneumonia. Clin Diagn Lab Immunol, 1996 Nov, 3(6), 717 - 21 Serum antibody response to outer membrane proteins of Moraxella (Branhamella) catarrhalis in patients with bronchopulmonary infection; Christensen JJ et al.; A Western blot (immunoblot) method for detecting antibodies against outer membrane protein (OMP) epitopes of Moraxella (Branhamella) catarrhalis was evaluated . Paired serum samples from patients suspected of M . catarrhalis (n = 38) and non-M . catarrhalis (n = 25) bronchopulmonary infection were examined for the presence of antibodies of the immunoglobulin M (IgM), IgG, and IgA classes to OMPs from M . catarrhalis by a gel electrophoresis-immunoperoxidase technique (Western blotting); sera from 40 healthy adult blood donors were also included . A significantly (P = 0.004) more frequent occurrence of IgM-class antibodies and/or an increase in the number of IgG-class antibodies against different M . catarrhalis OMPs from acute- to convalescent-phase serum samples was found for patients with M . catarrhalis (79%) than for patients without M . catarrhalis (40%) . IgM-class antibodies against OMPs of M . catarrhalis were found in acute- and/or convalescent-phase serum samples form 58% of patients with M . catarrhalis and 32% of patients without M . catarrhalis . Fifty percent of patients with M . catarrhalis and 16% of patients without M . catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgG-class antibodies directed against different OMPs . A total of 34% of patients with M . catarrhalis and 4% of patients without M . catarrhalis had, from acute- to convalescent-phase serum samples, an increased number of IgA-class antibodies directed against different OMPs . The present study indicates that M . catarrhalis is one of the bacteria involved in acute exacerbations of chronic bronchitis. Genetics, 1996 Nov, 144(3), 1063 - 73 Wolbachia infection and cytoplasmic incompatibility in Drosophila species; Bourtzis K et al.; Forty-one stocks from 30 Drosophila species were surveyed for Wolbachia infection using PCR technology . D . sechellia and two strains of D . auraria were found to be infected and were tested for the expression of cytoplasmic incompatibility, along with D . ananassae and D . melanogaster strains, which are already known to be infected . D . ananassae and D . melanogaster show levels of incompatibility up to 25%, while D . auraria and D . sechellia exhibit levels of egg mortality approximately 60% . A dot-blot assay using the dnaA sequence as probe was developed to assess the infection levels in individual males that were used in incompatibility crosses . A positive correlation between bacterial density and cytoplasmic incompatibility was observed . The stocks examined can be clustered into at least two groups, depending on the levels of infection relative to the degree of cytoplasmic incompatibility exhibited . One group, containing D . simulans Hawaii, D . sechellia, and D . auraria, exhibits high levels of cytoplasmic incompatibility relative to levels of infection; all the other species and D . simulans Riverside exhibit significantly lower levels of cytoplasmic incompatibility relative to levels of infection . These data show that, in addition to bacterial density, bacterial and/ or host factors also affect the expression of cytoplasmic incompatibility. Appl Environ Microbiol, 1996 Nov, 62(11), 3929 - 32 A simplified PCR-based method for the detection of Renibacterium salmoninarum utilizing preparations of rainbow trout (Oncorhynchus mykiss, Walbaum) lymphocytes; McIntosh D et al.; A method for the detection of Renibacterium salmoninarum by PCR is described . A rapid, reliable procedure was developed for the extraction of DNA, which could be applied to infected kidney homogenates and head kidney lymphocyte preparations . The target for DNA amplification was a 376-bp region of the gene encoding the 57-kDa major surface antigen (MSA) . The PCR was specific for R . salmoninarum and allowed the detection of 10 to 100 cells of the pathogen . Use of the PCR for the examination of experimentally infected rainbow trout showed it to be as reliable as plate culture methods for the detection of R . salmoninarum in infected kidneys. J Clin Microbiol, 1996 Nov, 34(11), 2766 - 9 Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms; Blasi F et al.; Recent reports suggest an association between Chlamydia pneumoniae and Helicobacter pylori bacteria and atherosclerosis . We studied 51 patients (mean age, 68.3 years) who underwent abdominal aortic aneurysm surgery . For each patient we performed a microimmunofluorescence test for immunoglobulin G (IgG), IgA, and IgM antibodies to C . pneumoniae specific antigen (TW-183) . Anti-H . pylori antibodies were determined by means of an EIA-G test . Each aortic aneurysm surgical specimen was sampled into multiple sections of 0.3 cm2 each and frozen at -20 degrees C . Two samples of each aneurysm were used for a nested PCR with two sets of C . pneumoniae and two sets of H . pylori specific primers . Specimens were treated with a solution containing 20 mM Tris-HCl, Tween 20-Nonidet P-40 (0.5% {vol/vol} each), and 100 micrograms of proteinase K per ml and incubated at 60 degrees C for 1 h and at 98 degrees C for 10 min . DNA was extracted twice with phenol-chloroform-isoamylic alcohol and precipitated with sodium acetate-ethanol by standard methods . Forty-one patients were seropositive for C . pneumoniae with past-infection patterns in 32 patients (16 < or = IgG < 512; 32 < or = IgA < 256) and high antibody titers in 9 patients (IgG > or = 512) . In 26 of 51 patients, C . pneumoniae DNA was detected in aortic aneurysm plaque specimens . Of these patients, 23 had a serologic past-infection pattern, 2 had an acute reinfection pattern, and 1 was seronegative . Forty-seven of 51 patients were seropositive for H . pylori . In all cases PCR showed no evidence of H . pylori presence in plaque specimens . This study provides data on a possible C . pneumoniae involvement in the pathogenesis of aortic aneurysm and additional evidence for an association between this agent and atherosclerosis . Conversely, notwithstanding a high H . pylori seroprevalence observed, our results tend to rule out the possibility of a direct involvement of H . pylori in atherosclerosis. J Clin Microbiol, 1996 Nov, 34(11), 2674 - 8 Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis; Tran SD et al.; Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis . However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive . A modified multiplex PCR was developed to address that question . Our method uses two species-specific forward primers in combination with a single reverse primer . These primers target variable and conserved regions of the 16S rRNA gene . Sensitivity was determined by testing serial dilutions of A . actinomycetemcomitans and P . gingivalis cells . Primer specificity was tested against (i) six A . actinomycetemcomitans strains and four P . gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects . The multiplex PCR had a lower limit of detection of 2 A . actinomycetemcomitans and 30 P . gingivalis cells . Species-specific amplicons were obtained for all A . actinomycetemcomitans and P . gingivalis strains tested and did not occur with seven other bacterial species unless A . actinomycetemcomitans and P . gingivalis were added . Neither target species was detected in supragingival plaque; A . actinomycetemcomitans was detected in one subgingival specimen, and P . gingivalis was detected in another . When plaque samples were spiked with 10 A . actinomycetemcomitans cells and 100 P . gingivalis cells, species-specific amplicons were detected . These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A . actinomycetemcomitans and P . gingivalis . This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens. Endocrinology, 1996 Nov, 137(11), 4637 - 43 Transfer of thyroiditis, with syngeneic spleen cells sensitized with the human thyrotropin receptor, to naive BALB/c and NOD mice; Costagliola S et al.; In previous studies we have induced TSH binding-inhibiting Igs and thyroiditis in BALB/c mice and thyroiditis alone in NOD mice immunized with the extracellular domain of the human TSH receptor produced as a maltose-binding protein fusion in bacteria (MBP-ECD) . In this study, our aim was to determine whether thyroiditis can be transferred to syngeneic naive recipients with in vivo and in vitro primed spleen cells . Groups of 6-week-old female BALB/c and NOD mice were immunized ip with MBP-ECD in an adjuvant of alum plus attenuated Bordetella pertussis toxin, on days 0 (100 micrograms), 14, 28, and 35 (50 micrograms) . These mice (in vivo primed) and nontreated age- and sex-matched controls were killed on day 43, and their spleens and thyroids were removed, the latter to verify the induction of thyroiditis in the antigen-treated mice . Splenocytes were disrupted mechanically and cultured at 3 x 10(6)/ml in RPMI supplemented with 20 micrograms/ml MBP-ECD for 48-64 h . After this in vitro priming, some of the splenocytes received no further treatment, but a portion was fractionated into a CD4+-enriched population . Groups of 6-week-old female BALB/c and NOD mice were immunized into the tail vein with 100-200 microliters PBS containing approximately 10(5)-10(7) unfractionated T cells (both in vivo primed and not) and CD4+-enriched (in vivo primed) splenocyte populations . The animals were killed 16 days later, and their thyroids were examined histologically and by immunohistochemistry . In addition, levels of antibody to the MBP-ECD priming antigen were assessed by enzyme-linked immunosorbent assay in the antigen- and spleen-treated mice . In the donor animals, in vivo priming resulted in an extensive lymphocytic infiltration of the thyroids in both BALB/c and NOD mice and follicular destruction in the latter . There was no evidence of thyroiditis in all 9 BALB/c mice and all 4 NOD mice who received unfractionated T cells from mice that had not been primed in vivo . In contrast, transfer of MBP-ECD in vivo primed unfractionated T cells resulted in thyroiditis in 9 of 13 BALB/c mice and 5 of 6 NOD mice; similarly, the equivalent CD4+-enriched population produced extensive thyroiditis in 2 of 3 BALB/c mice and all three NOD mice . The most striking difference between the antigen- and spleen-treated mice was in the quantity of the infiltrate, which was much greater in the latter and extended throughout the thyroid glands of these animals . In common with mice treated directly with the MBP-ECD antigen, the infiltrates of both BALB/c and NOD recipient mice contained large numbers of activated T cells expressing the receptor for interleukin-2, and macrophages and dendritic cells were plentiful, particularly in the BALB/c mice, in which B cells and interleukin-10-positive T cells were also present . The most abundant infiltrates, containing numerous CD8+ T cells and follicular destruction, were observed in NOD mice receiving primed unfractionated T cells or CD4+-enriched T cells . In contrast to the donors, none of the recipient animals had circulating antibodies to the MBP-ECD antigen . In conclusion, we have shown that it is possible to transfer thyroiditis with spleen cells from mice primed in vivo with a human TSH receptor preparation . Furthermore, the thyroiditogenic activity appears to reside in the CD4+ population. Infect Immun, 1996 Nov, 64(11), 4534 - 41 Immune responses and resistance to brucellosis in mice vaccinated orally with Brucella abortus RB51; Stevens MG et al.; Immune responses and resistance to infection with Brucella abortus 2308 (S2308) were measured in mice following oral or intraperitoneal (i.p.) vaccination with strain RB51 (SRB51) . Bacteria persisted in the parotid lymph node for 4 weeks following oral vaccination of mice with 5 x 10(8) or 5 x 10(6) CFU of SRB51 . Bacteria did not appear in the spleen during 12 weeks after oral vaccination, whereas they did appear in the spleen for 8 weeks following i.p . vaccination of mice with SRB51 (5 x 10(8) or 5 x 10(6) CFU) . Increased resistance to S2308 infection occurred at 12 to 20 weeks in mice vaccinated i.p . with SRB51 (5 x 10(8) or 5 x 10(6) CFU) but occurred at 12 weeks only in mice vaccinated orally with SRB51 (5 x 10(8) CFU) . Oral SRB51 vaccination induced lower levels of antibodies to the surface antigens of intact SRB51 bacteria than did i.p . vaccination . However, neither route of vaccination induced anamnestic antibody responses to the surface antigens of intact S2308 bacteria after challenge infection of the vaccinated mice with S2308 . Mice vaccinated orally with SRB51 and challenged with S2308 at 12 to 20 weeks had lower and less persistent spleen cell proliferation and production of gamma interferon in response to S2308 and certain immunodominant S2308 proteins (32 to < or = 18 kDa) than did mice vaccinated i.p . with SRB51 . However, mice vaccinated orally or i.p . with SRB51 and challenged with S2308 had similar spleen cell tumor necrosis factor alpha production . These results indicate that oral vaccination of mice with SRB51 was effective in inducing protective immunity to S2308 infection, although the immunity was lower and less persistent than that induced by i.p . vaccination . The lower protective immunity induced by oral vaccination may have resulted from lower and less persistent cell-mediated immunity and gamma interferon production in response to S2308 and S2308 proteins. Biophys Chem, 1996 Oct 30, 61(2-3), 73 - 84 Attractive adsorbate interaction in biological surface reactions; Nygren H; Recent studies on ferritin adsorption have revealed strong adsorbate interactions during adsorption (H . Nygren, Biophys . J., 65 (1993) 1508) . In the present study, efforts were made to quantify the attractive lateral adsorbate interaction and to describe the effect of such interactions on the time- and concentration dependence of biological surface reactions . Attractive forces between ligands increase the time that they are considered neighbours to other ligands . Thus, the inter-ligand interaction effects their distribution and this can be measured experimentally by the pair correlation function g(r) . The attractive lateral interaction was shown to affect the surface adsorption of ferritin, virus particles and bacteria, but played a minor role in the binding of antibodies to surface-immobilised antigen. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12570 - 4 Light-mediated retinoic acid production; McCaffery P et al.; Retinoids serve two main functions in biology: retinaldehyde forms the chromophore bound to opsins, and retinoic acid (RA) is the activating ligand of transcription factors . These two functions are linked in the vertebrate eye: we describe here that illumination of the retina results in an increase in RA synthesis, as detected with a RA bioassay and by HPLC . The synthesis is mediated by retinaldehyde dehydrogenases which convert some of the chromophore all-trans retinaldehyde, released from bleached rhodopsin, into RA . As the eye contains high levels of retinaldehyde dehydrogenases, and as the oxidation of retinaldehyde is an irreversible reaction, RA production has to be considered an unavoidable by-product of light . Through RA synthesis, light can thus directly influence gene transcription in the eye, which provides a plausible mechanism for light effects that cannot be explained by electric activity . Whereas the function of retinaldehyde as chromophore is conserved from bacteria to mammals, RA-mediated transcription is fully evolved only in vertebrates . Invertebrates differ from vertebrates in the mechanism of chromophore regeneration: while in the invertebrate visual cycle the chromophore remains bound, it is released as free all-trans retinaldehyde from illuminated vertebrate rhodopsin . RA synthesis occurring as corollary of dark regeneration in the vertebrate visual cycle may have given rise to the expansion of RA-mediated transcriptional regulation. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12142 - 9 The N-end rule: functions, mysteries, uses; Varshavsky A; The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue . Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria . In eukaryotes, the N-end rule pathway is a part of the ubiquitin system . I discuss the mechanisms and functions of this pathway, and consider its applications. Biochemistry, 1996 Oct 29, 35(43), 13858 - 64 Protein phosphatase type-1 and glycogen bind to a domain in the skeletal muscle regulatory subunit containing conserved hydrophobic sequence motif; Wu J et al.; This study identifies a 100-residue domain within the rabbit skeletal muscle regulatory subunit (PP1G) that binds both type-1 protein phosphatase (PP1C) and glycogen . An N-terminal portion of PP1G was cloned by RT-PCR, and different sized fragments were expressed in bacteria as glutathione S-transferase (GST) fusion proteins . A GST-PP1G fusion containing residues 51-240 bound both PPIC and glycogen, whereas GST alone or fusions containing residues 51-140 or 241-360 bound neither PP1C nor glycogen . The PPIC in whole cell lysates or partially purified PP1C from skeletal muscle, or a complex of PP1C-MCLR-biotin, all bound more effectively than Mn(2+)-activated, recombinant PP1C purified from bacteria . Binding was enhanced by increasing the ionic strength and was disrupted by ethylene glycol, consistent with hydrophobic interactions being critical for stable association . Phosphorylation of the GST-PP1G fusion by cAMP-dependent protein kinase prevented completely association of PP1C . This domain of PP1G, from residues 141-240, contains two sequence motifs of hydrophobic residues: Gx8FEKx10W and DxFxFxIxL, that are conserved among the known glycogen-binding PP1 regulatory subunits . These segments are predicted to form an alpha helix and a beta sheet, and we propose that they are the sites for association with PP1C and glycogen, respectively. J Biol Chem, 1996 Oct 25, 271(43), 26739 - 44 The binding protein for globular heads of complement C1q, gC1qR . Functional expression and characterization as a novel vitronectin binding factor; Lim BL et al.; A binding protein for the globular head domains of complement component C1q, designated gC1qR, recently described to be present on vascular and blood cells (Ghebrehiwet, B., Lim, B.-L., Peerschke, E . I . B., Willis, A . C., and Reid, K . B . M . (1994) J . Exp . Med . 179, 1809-1821 was expressed in recombinant form in bacteria to investigate its functional and structural properties . The recombinant gC1qR was found to be functional because tetramerization of the 24.3-kDa polypeptide occurred as described for the native protein, and the binding of the ligand C1q by recombinant gC1qR was indistinguishable from binding shown by gC1qR isolated from Raji cells . Recombinant gC1qR immobilized to microspheres was used to search for additional binding proteins unrelated to C1q . Surprisingly, it was found that vitronectin or complexes containing vitronectin were retained from plasma or serum, and subsequent analysis revealed the specific binding of the ternary vitronectin-thrombin-antithrombin complex to gC1qR . Because the thrombin-antithrombin complex was unable to interact with gC1qR, direct binding with vitronectin was investigated in a purified system . The heparin binding multimeric form of vitronectin but not the plasma form of vitronectin was found to bind specifically to gC1qR isolated from Raji cell membrane as well as to recombinant gC1qR . This interaction was saturable (KD approximately 20 nM) and inhibitable by glycosaminoglycans such as heparin but not by chondroitin sulfate . C1q and vitronectin did not compete with each other for binding to gC1qR, and both ligands seem to interact with different parts of the gC1qR because a truncated version of recombinant gC1qR lacking the N-terminal 22-amino acid portion hardly interacted with vitronectin but bound C1q as well as the intact gC1qR . These findings establish gC1qR as a novel vitronectin-binding protein that may participate in the clearance of vitronectin-containing complexes or opsonized particles or cooperate with vitronectin in the inhibition of complement-mediated cytolysis. J Biol Chem, 1996 Oct 18, 271(42), 26057 - 61 Manganese transport in the cyanobacterium Synechocystis sp . PCC 6803; Bartsevich VV et al.; We have inactivated the genes encoding components of MntABC, an ABC (ATP binding cassette) transporter system for manganese in the cyanobacterium Synechocystis sp . PCC 6803 . The growth rates of these mutant strains were significantly lower in a manganese-deficient medium and were restored to near normal levels upon addition of micromolar concentrations of Mn2+, indicating the presence of a second transport system for manganese in this organism . 54Mn2+ uptake experiments indicated that the MntABC transporter was induced under manganese starvation conditions, whereas the second transporter system was induced in the presence of micromolar levels of manganese . Both of these systems were nonfunctional at low temperatures and could transport trace levels of 54Mn2+, reflecting high affinity active transport . The initial rates of Mn2+ uptake for cells grown with or without manganese exhibited biphasic saturation kinetics, suggesting that Mn2+ can also be accumulated by a low affinity system in these bacteria . The kinetic parameters for the MntABC transporter system are Km = 1-3 microM and Vmax = 3-8 pmol/min.10(8) cells . Accumulation of manganese by this system was competitively inhibited by Cd2+ (Ki = 4-8 microM), Co2+ and Zn2+ (Ki = 8-15 microM) . In contrast, the second high affinity system was highly specific for manganese and was not inhibited by any tested metal ion . We have also demonstrated that in this organism, photosynthetic electron transport is necessary for optimal rates of manganese uptake. Gene, 1996 Oct 17, 176(1-2), 155 - 62 Sequence analysis of the omp2 region of Chlamydia psittaci strain GPIC: structural and functional implications; Hsia RC et al.; The nucleotide sequence of a 3.1-kb genomic DNA fragment carrying the omp3, omp2 and srp gene homologs from Chlamydia psittaci strain GPIC was determined . A comparative analysis of the GPIC sequence with other chlamydial omp2-linked sequences reveals highly conserved omp3 and omp2 upstream sequences across species, suggesting a unified mechanism of transcription regulation . In contrast, the omp2-srp intergenic segment, which encompasses hypothetical srp transcriptional initiation sites, is relatively less conserved in length and in sequence . Examination of the predicted translation products reveals a high degree of homology within Omp3 and Omp2 across species, with the notable exception of the N-terminal fifth of Omp2 . Although the latter segment displays relatively high interspecies sequence variation, it includes a smaller segment, whose high positive charge density is conserved across species, suggesting a conserved structure/function . In contrast to Omp2 and Omp3, a comparative analysis of the predicted amino acid (aa) sequence of the srp product reveals high homology within species, but relatively little across species . A 38-aa segment near the C-terminus of Srp, whose sequence is 64% identical between C . psittaci GPIC and C . trachomatis, is partially truncated in C . psittaci 6BC. Arch Microbiol, 1996 Oct 17, 166(4), 211 - 23 Anoxygenic phototrophy across the phylogenetic spectrum: current understanding and future perspectives Stackebrandt E, Rainey FA, Ward-Rainey N. The phylogenetic heterogeneity of anoxygenic phototrophic bacteria has been revealed by 16S rRNA sequence analysis, the results of which have led to extensive taxonomic rearrangements within previously defined taxa of phototrophs and stimulated interest in this group of organisms . Anoxygenic photosynthetic bacteria can be found within 4 of the 12 phylogenetic lineages, and in some cases are highly related to non-photosynthetic members of these groups . The largest number of phototrophs are found in the class Proteobacteria . Comparative phylogenetic analysis using 23S rDNA sequences generally supports the topology obtained from 16S rDNA sequences . The photosynthetic reaction centers are conserved in all photosynthetic bacteria, and are of two types . One is shared by the Proteobacteria and Chloroflexus aurantiacus and is similar to Photosystem II of cyanobacteria, while heliobacteria and Chlorobium and relatives possess a reaction center similar to the cyanobacterial Photosystem I . These similarities are supported by sequence analysis of core reaction center peptides, but contradict phylogenies reconstructed from rRNA sequence analysis . Genome analysis by means of physical mapping has been performed for only three species of anoxygenic phototrophs . Some conservation of operon structure and gene sequence has been found within the Proteobacteria, but does not extend to other phototrophs. Biochem J, 1996 Oct 15, 319 ( Pt 2), 499 - 506 Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2; Chion CK et al.; Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography . Both components were required for the degradation of epoxypropane and were purified further . Component A was apparently homohexameric with a subunit M(r) of about 44,000, and possessed NAD(+)-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity . It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide(NEM)and p-chloromercuribenzoate . Component B was homodimeric with a subunit M(r), of 62,170 and contained 2 mol.mol-1 FAD . It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate . The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane . NADPH was found to replace the need for a low-M(r), fraction in epoxypropane degradation assays containing components A and B and NAD+ . The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively . A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed. Gene, 1996 Oct 10, 175(1-2), 1 - 5 Synthesis and expression of genes encoding putative insect neuropeptide precursors in tobacco; Rao R et al.; Neuropeptides are the key molecules in a multiplicity of physiological processes and their use in pest control has recently been suggested . Most neuropeptides are produced in the form of a precursor that is cleaved by proteolysis to yield various biologically active peptides . To mimic this structure, a method has been developed for synthesizing genes that code for putative polyneuropeptide precursors . As a model neuropeptide, the 5-amino-acid proctolin, one of the best studied invertebrate neuropeptides, functioning both as a visceral and a skeletal neuromuscular transmitter, was chosen . The synthetic gene was introduced into bacteria and tobacco plants, where it was efficiently transcribed . We present our results as a possible approach for the expression, in a variety of organisms, of synthetic genes coding for a wide repertoire of insect neuropeptides. Cell Immunol, 1996 Oct 10, 173(1), 155 - 61 Level of myelopoiesis in the bone marrow is influenced by intestinal flora; Tada T et al.; Mice were orally given kanamycin (50 mg/day/ mouse) for 1 or 2 weeks . Almost all bacteria in the intestine were eliminated within a week . In parallel with this elimination, the level of granulocytes in the bo