Pac Symp Biocomput . 1998;:485-96. Function driven protein evolution . A possible proto-protein for the RNA-binding proteins; Fetrow JS et al.; We introduce a hypothesis that present day proteins evolved from "proto-proteins," small 15-20 residue peptides with some elements of secondary structure and primitive function . Increasingly stable and functional proteins arose by adding structural elements to produce the small domains or protein modules that we would recognize today . From this point of view, the surprising similarities between small structural fragments of large proteins, that are usually taken as examples of convergent, function-driven evolution, are interpreted in exactly the opposite way--as traces of common evolutionary origin . As an example, a hypothetical evolutionary tree for two families of RNA binding proteins, the OB fold, a family of all beta proteins, and RBD fold, an alpha/beta protein family is presented . We argue that both protein families could have evolved from the same RNA-binding proto-protein, which had a form of beta-loop-beta RNA binding motif. Pac Symp Biocomput . 1998;:77-88. Qualitative analysis of gene networks; Thieffry D et al.; In this paper, we review the qualitative tools developed by our group for the analysis of regulatory networks . Focusing on the dynamical and biological roles of feedback circuits, this method can be applied in the context of both logical and differential formalisms . This approach already led to several interesting results about the relation between the network structure and the corresponding dynamical properties . In particular, it could be shown that at least one positive regulatory circuit is necessary to generate multistationarity (i.e., alternative states of gene expression), whereas at least one negative circuit is necessary to generate a stable oscillatory behavior . Applications to the analysis of complex gene networks, as well as to the synthesis of regulatory models to account for global expression data are discussed. J Pharmacokinet Biopharm, 1997 Dec, 25(6), 713 - 30 A pharmacokinetic-pharmacodynamic model of chemotherapy of human immunodeficiency virus infection that relates development of drug resistance to treatment intensity; Jackson RC; RNA viruses, including retroviruses, have mutation rates that are about 100 times higher than those of DNA viruses, bacteria, or eukaryotes, so that resistance to AIDS drugs emerges very rapidly . This has been shown to limit the effectiveness of the treatment of AIDS by reverse transcriptase inhibitors, such as zidovudine (AZT) and resistance to the new class of HIV aspartyl protease inhibitors has already been reported . The technique of pharmacokinetic-pharmacodynamic simulation has now been used to predict ways of delaying the development of resistance to these two classes of antiretroviral agents . A model is described that includes pharmacokinetic, pharmacodynamic, and cytokinetic equations, and expressions describing effects if the HIV on the immune system and destruction of virally infected cells by cellular immunity . The model predicted that the degree of viral drug resistance in relation to the sustainable blood level of drug would be the major determinant of response duration . Early treatment was consistently superior to late treatment, both with a drug that caused cumulative toxicity and with a drug that did not . Making reasonable assumptions about the likely degree of viral resistance, in conjunction with typical blood levels achievable for reverse transcriptase inhibitors or aspartyl protease inhibitors led to predicted response durations of several months to a few years, despite the rapid mutation rate of HIV . Preliminary studies of combination chemotherapy showed that predicted response durations were greater than for monotherapy, though less than the sum of responses to the individual drugs . Strategies for delaying the development of resistance include early treatment, combination chemotherapy, and developing novel agents with a high ratio of plasma level to antiviral efficacy. Nippon Ishinkin Gakkai Zasshi, 1998, 39(3), 121 - 2 Introduction: electron microscopy for fungal cell ultrastructure Kitajima Y. Ultrastructural morphology is a principal study for almost all natural science, i.e., an inevitable and fundamental study to elucidate structures, functions and differentiation of a given tissue, cell, or molecule as a most understandable visual form . The number of young scientists, however, who have been engaged in this field is very small recently, since this field appears to be too old when compared with modern molecular biology and it takes much longer time for the beginner to master the methology for electron microscopy (EM) . This symposium is designed for these young scientists and molecular biologists or biochemists, who are not so familiar to ultrastructural morphology, to better understand the applicability of EM, through new results and findings in ultrastructures of fungal cells and related organisms . EM includes several kinds of methods, which are shadowing EM, negative staining EM, ultrathin section EM, scanning EM, freeze-fracture EM, immuno-EM and diverse methods for staining the specimen . Shadowing EM and negative staining EM are suitable methods for the study molecular structures of proteins, and the former is prepared by shadowing with platinum palladium in a vacuum chamber, and the latter is a method to observe a relief prepared by dipping the sample in phosphotungsten solution . Freeze-fracture electron microscopy is suitable for the study of membrane plane ultrastructures, since it reveals a wide planar view of the membrane by splitting it along the hydrophobic membrane internal plane . Immunoelectron microscopy is an essential method for the study of intracellular localization of proteinous molecules . These methods will be introduced . This symposium will introduce new findings as for fungal cells, bacteria and protozoa obtained principally by using electron microscopy . These findings obtained through ultrastructures may provided a renewed knowledge of research approach from view points of ultrastructure. J Bacteriol, 1998 Aug, 180(16), 4258 - 69 Physiological control and regulation of the Rhodobacter capsulatus cbb operons; Paoli GC et al.; The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators . As a prelude to studies of cbb gene regulation in R . capsulatus, the nucleotide sequence of a 4,537-bp region, which included cbbRII, was determined . This region contained the following open reading frames: a partial pgm gene (encoding phosphoglucomutase) and a complete qor gene (encoding NADPH:quinone oxidoreductase), followed by cbbRII, cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase) . Physiological control of the CBB pathway and regulation of the R . capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs . Characterization of mutant strains revealed that either form I or form II ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS and cbbM genes, respectively, could support photoheterotrophic and autotrophic growth . A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium . Disruption of cbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one cbbP gene in R . capsulatus and that this gene is cotranscribed with cbbM . Analysis of RubisCO activity and synthesis in strains with disruptions in either cbbRI or cbbRII, and beta-galactosidase determinations from wild-type and mutant strains containing cbbIp- and cbbIIp-lacZ fusion constructs, indicated that the cbbI and cbbII operons of R . capsulatus are within separate CbbR regulons. Am J Physiol, 1998 Jun, 274(6 Pt 1), G992 - 6 H . pylori stimulates gastrin release from canine antral cells in primary culture; Lehmann FS et al.; Patients chronically infected with Helicobacter pylori are known to have hypergastrinemia . Previous studies have demonstrated the stimulation of gastrin from isolated G cells by monocytes and cytokines . The aim of this study was to determine if H . pylori can directly stimulate gastrin secretion . The secretion of gastrin from canine G cells in 48-h primary cultures was investigated using either live H . pylori bacteria or various bacterial extracts from three well-characterized strains . Whole bacterial sonic extracts and water-extracted surface proteins, but not PBS extracts, from strains 43579 (CagA+/VacA+), 60190 (CagA+/VacA+), and 60190:v1 (CagA+/VacA-) significantly stimulated gastrin release . Controls demonstrated that gastrin stimulation by the sonic extracts was not due to a direct toxic effect on G cells . We conclude that H . pylori produces a soluble factor(s), which can directly stimulate gastrin release in enriched canine G cell cultures . This stimulatory effect may play an important role in the H . pylori-associated hypergastrinemia and subsequent development of peptic ulcer disease. J Neurooncol, 1998 Jun-Jul, 38(2-3), 97 - 102 Cellular and molecular mechanisms of metastasis as applied to carcinomatous meningitis; Mareel M et al.; Cancer cells as well as bacteria metastasize to the subarachnoidal space (SAS) causing meningitis . Primary brain tumors, although not forming distant metastases, disseminate via the cerebrospinal fluid and occupy the meninges . The multistep process of cancer or bacterial dissemination is regulated through molecular crosstalk between invaders and host cells . Such crosstalks establish invasion-promoter and invasion-suppressor complexes . In carcinomatous and bacterial meningitis, the participation of host cells is prominent since leukocytes and inflammatory cytokines are the major determinants of malignancy . We propose a model in which bacterial breakdown products activate endothelial cells, a process leading to leukocyte extravasation . This initiates a cascade of inflammatory processes opening up the blood cerebrospinal fluid barrier and producing access for new invaders. Trends Cell Biol, 1998 Apr, 8(4), 133 - 7 Atomic structures of tubulin and FtsZ; Erickson HP; The recently published atomic structures of tubulin and FtsZ are a research milestone . The N-terminal GTP-binding domains of tubulin and FtsZ are virtually identical in structure, as expected from the substantial sequence identity . Sequence identity is absent from the C-terminal domains, but they also have virtually identical structures . A surprising finding is that the N-terminal GTP-binding domain is structurally homologous to that of Ras and other G proteins, despite the completely different GTP-binding sequence motifs . This article discusses these findings and the molecular mechanisms that can now be addressed with the atomic structures. Parasitology, 1998, 116 Suppl, S65 - 72 Displaced tick-parasite interactions at the host interface; Nuttall PA; Reciprocal interactions of parasites transmitted by blood-sucking arthropod vectors have been studied primarily at the parasite-host and parasite-vector interface . The third component of this parasite triangle, the vector-host interface, has been largely ignored . Now there is growing realization that reciprocal interactions between arthropod vectors and their vertebrate hosts play a pivotal role in the survival of arthropod-borne viruses, bacteria, and protozoa . The vector-host interface is the site where the haematophagous arthropods feeds . To obtain a blood meal, the vector must overcome the host's inflammatory, haemostatic, and immune responses . This problem is greatest for ixodid ticks which may imbibe as much as 15 ml blood whilst continuously attached to their host for 10 days or more . To feed successfully, the interface between tick and host becomes a battle between the host's mechanisms for combating the tick and the tick's armoury of bioactive proteins and other chemicals which it secrets, via saliva, into the feeding lesion formed in the host's skin . Parasites entering this battlefield encounter a privileged site in their vertebrate host that has been profoundly modified by the pharmacological activities of their vector's saliva . For example, ticks suppress natural killer cells and interferons, both of which have potent antiviral activities . Not surprisingly, vector-bone parasites exploit the immunomodulated feeding site to promote their transmission and infection . Certain tick-bone viruses are so successful at this that they are transmitted from one infected tick, through the vertebrate host to a co-feeding uninfected tick, without a detectable viraemia (virus circulating in the host's blood), and with no untoward effect on the host . When such viruses do have an adverse effect on the host, they may impede their vectors' feeding . Thus important interactions between ticks and tick-borne parasites are displaced to the interface with their vertebrate host-the skin site of blood-feeding and infection. J Biol Chem, 1998 Aug 14, 273(33), 21374 - 9 The membrane proximal cytokine receptor domain of the human interleukin-6 receptor is sufficient for ligand binding but not for gp130 association; Ozbek S et al.; Interleukin-6 (IL-6) belongs to the family of the "four-helix bundle" cytokines . The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains . Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif . On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6.IL-6R associates with the signal transducing protein gp130 . The IL-6R consists of three extracellular domains . The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation . Here we have investigated the properties and functional role of the third membrane proximal domain . The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding . When complexed with IL-6, however, it fails to associate with the gp130 protein . Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling. Pathobiology, 1998, 66(3-4), 159 - 64 Gastrointestinal pathology in rhesus monkeys with experimental SIV infection; Kaup F et al.; The updated results of current pathomorphological investigations in SIV-infected rhesus monkeys (Macaca mulatta) are summarized . After experimental infection with several SIVmac251 subtypes and various vaccination trails, 147 rhesus monkeys were morphologically examined until now . The pathology of the gastrointestinal tract in SIV-infected animals resembled those of human cases with HIV and AIDS . Alterations were considered to be primary SIV-induced (SIV enteropathy, giant cell disease) or secondary caused by opportunistic agents . Typical secondary gastrointestinal opportunistic infectious agents were parasites (Cryptosporidium sp., Trichuris sp., Trichomonas sp., Spironucleus sp.), viruses (cytomegalovirus, adenovirus) and bacteria (Mycobacterium simiae) . Five animals developed malignant lymphomas involving the intestinal tract . The present observations revealed that SIV infection of rhesus monkeys provide an excellent model for studies on the pathogenesis of HIV in man. Parasitol Res, 1998 Jul, 84(7), 583 - 9 Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa); Freyer B et al.; A monoclonal antibody (2F4) directed against a 32-kDa dense-granule antigen of Sarcocystis muris cyst merozoites (bradyzoites) was used to screen a lambda ZAP cDNA expression library . A clone with an insert of 1.4 kb in length (DG 32/1) was isolated . A fusion protein derived from bacteria harbouring the recombinant plasmid DG 32/1 reacted with monoclonal antibody (mAb) 2F4 . Southern blot hybridization suggests that the gene is present as a single copy . On Northern blots, a single mRNA species of 1.8 kb was detected by a cDNA-derived probe . In addition, we isolated a full-length clone (DG 32/PH1) by screening the cDNA library with a non-radioactive-labelled cDNA probe . The nucleotide sequence of DG 32/PH1 comprises 1.57 kb . It contains an open reading frame of 882 bp with a coding capacity of approximately 32 kDa . The hypothetical polypeptide consists of a putative N-terminal signal peptide and the mature protein sequence . The occurrence of an N-terminal signal sequence is consistent with the observation that the 32-kDa protein of S . muris is secreted from the dense granules. J Vet Dent, 1994 Oct, 11(3), 106 - 9 Acute and chronic alveolitis/osteomyelitis ("lumpy jaw") in small exotic ruminants; Wiggs RB et al.; Tooth-related abscesses in small captive ruminants are most likely foreign body-induced periodontic-endodontic lesions . The instigating cause may be abnormal texture of dietary material . Bacteria, though probably not the initiating cause, significantly contribute to pathology and morbidity . Extraction of severely affected teeth is an effective although sometimes challenging mode of treatment . Periodically complications may arise, which vary in difficulty of treatment . Prevention may be the alteration of the major forage component of the diet to textures that are less coarse and stemmy, and the alleviation of any crowding and sanitation problems. Dtsch Tierarztl Wochenschr, 1998 Jun, 105(6), 225 - 34 {Health effects of airborne pollutants, particularly in swine confinement stalls, from the viewpoint of occupational medicine}; Nowak D; From the point of view of occupational respiratory medicine, an overview on potential health effects of airborne pollutants particularly in swine confinement houses is presented . Airway diseases are the most frequent occupational disorders among farmers in many countries around the world including Germany . Due to various methodological reasons, epidemiological studies in farming populations are more difficult to perform than among non-farmers . Major constituents of swine confinement dust include bacteria, endotoxin, mites, fungal spores, and animal dander . Gaseous pollutants include ammonia, methane, and hydrogen sulfide . In a variety of cross-sectional studies, high prevalences of respiratory symptoms and non-obstructive (and obstructive) bronchitis and Organic Dust Toxic Syndrome have been reported in pig farmers . Nasal and bronchial provocation challenges with swine confinement dust include influx of neutrophils and other inflammatory cells as well as mediators . In cross-sectional and longitudinal studies, endotoxin turns out as the probably most relevant parameter associated with lung function impairment . Further studies are clearly needed focusing on the prognosis of non-obstructive bronchitis in swine farmers and on health effects of reducing airborne contaminants in swine confinement houses. Front Biosci, 1998 Aug 05, 3, e141 - 8 Immunopathogenesis of Mycobacterium avium infection; Cooper AM et al.; One of the most obvious problems one perceives when working with Mycobacterium avium isolates is the vast array of phenotypes expressed with regard to colonial morphotype, serovar and particularly virulence . Thus whenever experimental data derived from different MAC isolates is compared the variety of this group of mycobacteria must always be considered . Another issue of concern is the extrapolation of in vitro data to the in vivo disease . We have reported, in the past, that survival in murine macrophage culture does not always correlate with survival in vivo (23) . It is plausible therefore, that the pathways outlined in section 5.2 and figure 3 play a crucial role in the initiation of the innate immune response in general and that there are components of this response which are not expressed by IFN-gamma activated macrophages but which are necessary for bacterial control . In conclusion, we suggest that the initial control of MAC infection requires a healthy lung (or gut) architecture and that control by unactivated macrophages includes respiratory burst activity and also the sequestration of free iron away from the mycobacterial phagosome . Acquired immunity is important in controlling bacteria which have overcome the innate response and this control is mediated by cytokine activation of infected macrophages . Finally, we have described an animal model of infection in which uncontrolled bacterial growth occurs and in which lesions similar to those seen in AIDS patients develop. Protein Expr Purif, 1998 Aug, 13(3), 414 - 22 Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase; Bader B et al.; Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate . Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines . Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer . In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein . Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells . By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum . Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag . HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol . By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone . The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes . Protein Expr Purif, 1998 Aug, 13(3), 383 - 8 Prothymosin alpha is not found in yeast; Trumbore MW et al.; According to published accounts, prothymosin alpha exhibits high evolutionary conservation from yeast to man (Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A . FEBS Lett . 257, 247-250, 1989) . We report here our failure to find evidence for prothymosin alpha in yeast using three biochemical approaches: hybridization of yeast mRNA and genomic DNA with human prothymosin alpha coding region probes, performance of the polymerase chain reaction with yeast genomic template DNA and three sets of primers recognizing human prothymosin alpha coding region sequences, and isolation of yeast proteins essentially as described in the publication above . A survey of the Saccharomyces cerevisiae complete genome database using the program BLASTp verified our findings: there is no prothymosin alpha-homologue in yeast . Furthermore, DNA representing organisms from bacteria to amphibians also failed to hybridize with the same probes . Therefore, the presence of a prothymosin alpha gene in animals other than mammals is highly unlikely . Eur Biophys J, 1998, 27(4), 409 - 10 Sequence similarities of glyceraldehyde-3-phosphate dehydrogenases, phosphoglycerate kinases, and pyruvate kinases are species optimal temperature-dependent; Early CN et al.; Data are presented that suggest enzyme sequence similarities among species are not solely a function of their evolutionary relationship . It is demonstrated that sequence similarities of glyceraldehyde-3-phosphate dehydrogenases, phosphoglycerate kinases, and pyruvate kinases from yeast, bacteria, mammals and a bird possess a significant species optimal thriving temperature dependence that crosses through conventional phylogenetic divisions . It is therefore suggested that species which are distantly related evolutionarily may possess some degree of enzyme sequence similarity if they happen to thrive at near the same optimal temperature; conversely, organisms which are closely related evolutionarily but function at radically different temperatures will possess a sequence dissimilarity that may mask the close relatedness. Vestn Khir Im I I Grek, 1998, 157(2), 66 - 8 {Controlled opening of wounds with the apparatus of pin cutaneous fixation as a method of treatment in anaerobic infections of the lower limbs}; Chertov EA et al.; A method of postoperative treatment of the anaerobic infection of the lower extremity soft tissues is proposed which consists in the wide opening of the wounds with a simple device . Fixation of the extremity in the device is provided by the intracutaneously conducted and strained extension wires . Due to the effect of decompression of the tissues, gaping wounds and extension of the skin margins, there appear conditions for subsiding the inflammation, effective control, management of the wounds and closing of them by secondary sutures to form a linear cicatrix . The authors succeeded in shortening the time of preparing the wound for closure and obtaining better results. Curr Opin Biotechnol, 1998 Jun, 9(3), 288 - 91 Novel evolutionary histories and adaptive features of proteins from hyperthermophiles Robb FT, Maeder DL. The hyperthermophiles include both bacteria and archaea, although the majority of isolates growing above 100 degreesC are archaea . Newly described adaptive features of hyperthermophiles include proteins stable to 200 degreesC, nucleosomes, chaperonins and high-capacity DNA modifying enzymes . The ongoing release of genomic sequence data from hyperthermophiles will continue to accelerate the discovery of novel proteins. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1998 Jul, 86(1), 98 - 103 A histopathologic study of direct pulp-capping with adhesive resins; Olmez A et al.; OBJECTIVE: The purpose of this study was to evaluate the histologic pulp responses of Optibond and Syntac adhesive resin systems placed directly on exposed pulp tissues . STUDY DESIGN: Class V facial cavities with pulpal exposures were prepared in dogs . After acid etching of enamel margins, the cavities were restored with a composite resin after pulp-capping with one of the dentin bonding agents . The remaining exposures were capped with calcium hydroxide and amalgam as controls . The animals were killed after 7, 21, and 90 days and the pulps were evaluated histologically . Statistical analysis was carried out with the chi-square test . RESULTS: There was no statistically significant difference with respect to inflammatory cell response, fibrosis, bleeding, or bacterial staining criteria over the time intervals of evaluation among the Optibond, Syntac, and calcium hydroxide groups . New dentin formation was also observed for all of the groups at the end of 90 days . CONCLUSION: The results of direct pulp-capping with a dentinal adhesive and composite resin appear promising but further in vivo studies are recommended. Vet Q, 1998, 20 Suppl 3, S49 - 52 Food allergy, coeliac disease and chronic inflammatory bowel disease in man; Pena AS et al.; It is often stated that the gastrointestinal tract has a limited number of responses to pathogens . Entirely different agents can produce a similar histopathological reaction . However, the expression of the disease in man is very heterogeneous, it varies with the age of the subject and is to a certain extent genetically determined . For example, food allergy is frequent in childhood and not common in adulthood . The intestinal mucosa in the child with cows milk allergy shows a 'flat' mucosa, which may be indistinguishable of that observed in gluten sensitive enteropathy or coeliac disease . Subjects with other forms of food allergy may have a morphologically normal small intestinal mucosa, occasionally with increased IgE plasma cells and often only characterised by an increased intestinal permeability . An abnormal intestinal permeability is one of the hallmarks of an inflamed gut, however, subjects with a latent form of coeliac disease have an abnormal permeability only without overt signs of inflammation . Recently, it has become clear that what determines the characteristics of the intestinal inflammatory response is dependent on the cytokines involved during the response and this seems to be the same in the stomach, the small intestine and the colon . A so-called Th1 response, with an increased production of IFN-gamma, TNF-alpha and other pro-inflammatory cytokines, occurs in the stomach when infected by Helicobacter pylori, in the small intestine when the subject with coeliac disease consumes normal bread and during the active phases of Crohn's disease . A Th2 response is characteristic of the allergic subject and there is some evidence that it is the predominant response in subjects with ulcerative colitis . We still do not know the fine-tuning of the cytokine response but IL-12 appears to be a key cytokine in polarising the response to a Th1 type . More recently it has become clear that the intestinal mucosa has a unique subset of CD4+ T cells that secrete TGF-beta (Th3 cells) that provide help for IgA . These cells have downregulatory properties for Th1 cells and therefore play an important role in the active suppression of oral tolerance and IgE response . What determines that an individual develops one of these diseases? It is now clear that these different pathological entities are multifactorial . Different environmental factors and a complex genetic predisposition where more that one gene and more than one chromosome are involved . The extent and severity of the inflammatory response depends on the genetic diversity of the bacteria or the amount of the antigen on the one hand and on the genetic constitution of the host on the other . The abnormal immune response in the human gut is predominantly a Th1-like inflammatory response . This can be elicited by bacteria, peptides, possibly the bacterial flora and some viruses . The recent findings in the pathogenesis of the intestinal inflammatory response will probably alter the therapy of the future. Acta Anaesthesiol Scand, 1998 Jul, 42(6), 664 - 9 No inhibition of gastro-intestinal propulsion after propofol- or propofol/ketamine-N2O/O2 anaesthesia . A comparison of gastro-caecal transit after isoflurane anaesthesia; Freye E et al.; BACKGROUND: Gastrointestinal motility may be considerably reduced by anaesthesia and or surgery resulting in postoperative ileus . Inhibition of propulsive gut motility is especially marked after an opioid-based technique . Little, however, is known of the gastrointestinal effects of the hypnotic propofol when given continuously over a longer period of time, which is the case in total intravenous anaesthesia (TIVA) and in intensive care sedation . We therefore set out to study the effects of a propofol-based nitrous oxide/oxygen anaesthesia (group PO) on gastro-caecal transit time . The results were compared with a propofol-ketamine technique (group PK) and an isoflurane-based anaesthesia (group I; each group n = 20) . METHODS: Gastro-caecal transit was determined by measurement of endexpiratory hydrogen concentration (ppm) . Following gastral installation of lactulose at the end of the operation, the disaccharide was degraded by bacteria in the caecum, resulting in the liberation of hydrogen which was expired . A 100% increase in endexpiratory hydrogen concentration compared to the preinduction period was considered the end-point of gastro-caecal transit . RESULTS: There was no significant difference with regard to gastro-caecal transit in the three groups of patients . In the propofol group mean gastro-caecal transit was 119 (+/- 50.6 SD) min, in the propofol-ketamine group it was 147 (+/- 57.4 SD) min, and in the isoflurane group transit time was 122 (+/- 48.6 SD) min . CONCLUSION: The data suggest that propofol, even when given as a continuous infusion, does not alter gastrointestinal tract motility more than a standard isoflurane anaesthesia . The data may be particularly relevant to patients who are likely to develop postoperative ileus . They also suggest that in an ICU setting propofol does not alter gut motility more than a sedation technique with the analgesic ketamine. Am J Physiol, 1998 Jul, 275(1 Pt 1), L1 - 13 Surfactant protein A and surfactant protein D in health and disease; Mason RJ et al.; Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung . Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin . Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules . SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin . Initially, most research interest focused on its role in surfactant homeostasis . Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells . SP-D is much less involved with the surfactant system . SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin . Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis . In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease . The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo. Appl Environ Microbiol, 1998 Aug, 64(8), 2937 - 42 Enzyme-linked immunofiltration assay To estimate attachment of thiobacilli to pyrite Dziurla MA, Achouak W, Lam BT, Heulin T, Berthelin J. An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals . This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates . The polyclonal antiserum used in this study was raised against T . ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus . This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10(4) bacteria were detected per well of the microtiter plate) . The mean value of bacterial attachment has been estimated to be about 10(5) bacteria mg-1 of pyrite at a particle size of 56 to 65 &mgr;m . The geometric coverage ratio of pyrite by T . ferrooxidans ranged from 0.25 to 2.25% . This suggests an attachment of T . ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties . ELIFA was shown to be compatible with the measurement of variable levels of adhesion . Therefore, this method may be used to establish adhesion isotherms of T . ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces. FEBS Lett, 1998 Jul 3, 430(3), 181 - 5 The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase; Zhou NY et al.; The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced . The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (> 60% sequence similarity) and methane monooxygenase (> 40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present . Secondary structure prediction based on the amino acid sequence showed that the predominantly alpha-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase alpha-subunit . Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases. J Antimicrob Chemother, 1998 Jun, 41 Suppl D, 81 - 93 Viral infections in neutropenia--current problems and chemotherapeutic control; Wood MJ; The risk of infection in immunocompromised patients is determined by the nature, degree and duration of the immunosuppressive disease or therapy . Although neutropenia is clearly related to an increased risk of infection, these infections are typically caused by bacteria and fungal pathogens rather than by viruses . Viral infections are predominantly associated with defects in cellular immune function and might not be expected to cause problems in patients whose primary disease is accompanied by neutropenia . The net state of the patient's host-defence mechanisms is, however, a complex interplay between a number of factors, primary among which are the underlying disease and the nature of the therapy being given . In certain periods of neutropenia, therefore, particularly that occurring early after bone marrow transplantation, viral infections are commonly seen . The viruses responsible are chiefly the herpesviruses, both primary infections and reactivation, although other viruses are assuming recognized importance in this setting . This article provides a review of the infections that are encountered during the period of neutropenia in immunocompromised patients and the options available for chemotherapeutic management. Rev Laryngol Otol Rhinol (Bord), 1997, 118(5), 291 - 4 On the function of the mastoid cavity with emphasis on its role in middle ear defence; Radovic N et al.; The function of the mastoid cavity is still unknown . In the present article a theory of its main function is presented . The mastoid cavity is of vital importance in the non-immunological defence of the middle-ear . It is the most important site for the production of secretions that "wash" the middle-ear free of bacteria and viruses . The mucociliary system and the gravitation force, if the body is in erect position, transports the secretions through the two openings of the ear, the aditus and antrum and the ostia of the Eustachian tube, to be expelled into the epipharynx . Based on this theory pathological disorders of the ear are discussed as well as the effect of ventilation tubes on otosalpingitis and recurrent otitis media . Other plausible mastoid cavity functions are also discussed. Appl Environ Microbiol, 1998 Aug, 64(8), 2888 - 93 Detection of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Pleuroceridae: Juga spp.) from northern California; Barlough JE et al.; Ehrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae: Juga spp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic . Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA, groESL heat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembled Ehrlichia risticii, the causative agent of PHF . The minimum percentage of Juga spp . harboring the organism in the population studied was 3.5% (2 of 57 snails) . No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site . These data suggest that pleurocerid stream snails may play a role in the life cycle of E . risticii in northern California. Biol Chem, 1998 Jun, 379(6), 743 - 7 Molecular evolution of hydantoinases; May O et al.; The complete amino acid sequence of the hydantoinase from Arthrobacter aurescens DSM 3745 has been derived by automated Edman degradation . This is the first ever reported amino acid sequence of a non-ATP-dependent hydantoinase, which hydrolyzes 5'-monosubstituted hydantoin derivatives L-selectively . A homology search performed in protein and nucleic acid databases retrieved only distantly related proteins . All of these are members of the recently described protein superfamily of amidohydrolases related to ureases (Holm and Sander, Proteins 28: 72-82, 1997) . Phylogenetic analysis revealed that the novel hydantoinase forms a new branch separate from other hydantoin cleaving enzymes like dihydropyrimidinases (EC 3.5.2.2) and allantoinases (EC 3.5.2.5) . Our results suggests that the enzymes of this protein superfamily have evolved from a common ancestor and therefore are the product of divergent evolution . We show further that the enclosed gene families developed very early in evolution, probably prior to the formation of the three domains, Archaea, Eukarya and Bacteria . Hydantoinases related to ATP-dependent N-methylhydantoinases (EC 3.5.2.14) or 5-oxoprolinases (EC 3.5.2.9) do not belong to this superfamily. Adv Ren Replace Ther, 1998 Jul, 5(3), 185 - 93 Factors increasing severity of peritonitis in long-term peritoneal dialysis patients; Park MS; Peritonitis is the most frequent complication and a leading cause of discontinuation of peritoneal dialysis (PD) . Intact epithelial lining, sufficient blood flow, and adequate immunologic responses are vital to eradicate infection . In long-term PD, various pathological changes such as denudation of peritoneal mesothelial cells, duplication of submesothelial and/or capillary basement membranes, submesothelial fibrin deposit, and peritoneal fibrosis have been reported . Causes of these changes of the peritoneum are multifactorial . Commonly used dialysis solutions that are acidic, hypertonic, containing high concentrations of glucose and lactate, contaminated by glucose and/or plastic degradation products are not biocompatible and may induce chronic immune reactions in the peritoneal cavity . Long-term exposure of the peritoneum to dialysis solutions, the peritoneal catheter, and recurrent episodes of peritonitis all contribute to peritoneal injury . In addition, long-term exposure of peritoneal cells such as macrophages, mesothelial cells, and fibroblasts to dialysis solutions may also alter the normal immunologic reactions against bacteria . Peritoneal concentrations of opsonins such as Ig, complement, and protease are approximately 1% of the serum levels and far below the level sufficient to eradicate bacteria due to continuous peritoneal lavage and dilution with dialysis solutions . Furthermore, glycation of IgG induces chronic activation of macrophages and decreases normal opsonic activities against bacteria . Fibrin deposits, collagen accumulation, and cellular desert of the peritoneum observed in long-term peritoneal dialysis patients may serve as a safe shelter for bacteria from contact with inflammatory cells and opsonin and delay eradication of bacteria . In conclusion, peritonitis is often more severe in patients on long-term PD . In this setting, peritonitis needs special attention to prevent life-threatening infection and further damage of the peritoneum. J Inherit Metab Dis, 1998, 21 Suppl 1, 40 - 58 The biochemical and molecular spectrum of ornithine transcarbamylase deficiency; Tuchman M et al.; Ornithine transcarbamylase (OTCase) deficiency, the most common inherited urea cycle disorder, is transmitted as an X-linked trait . The clinical phenotype in affected males as well as heterozygous females shows a spectrum of severity ranging from neonatal hyperammonaemic coma to asymptomatic adults . The ornithine transcarbamylase enzyme is a trimer with three active sites per holoenzyme molecule, each of which is composed of an interdomain region of one polypeptide and a polar domain of the adjacent polypeptide . The OTC gene is located on the short arm of the X-chromosome and one of the two alleles undergoes inactivation in female cells . Approximately 140 mutations have been found in families affected with OTCase deficiency, most having their own 'private' mutation . Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions . Approximately 15% of mutations affect RNA splicing sites . The recurrent mutations are distributed equally among CpG dinucleotide hot spots . Generally, mutations causing neonatal disease affect amino acid residues that are 'buried' in the interior of the enzyme, especially around the active site, while those associated with late onset and milder phenotypes tend to be located on the surface of the protein . Very few mutations have been found in the sequence of the leader peptide, proportionally much fewer than in the sequence of the mature enzyme . Only few of the mutations have been expressed in bacteria or mammalian cells for the study of their deleterious mechanisms . Examples of expressed mutations include R277W and R277Q associated with late-onset disease, which markedly increase the Km for ornithine, shift the pH optimum to more alkaline and decrease the thermal stability of the purified mutant enzyme . R141Q (neonatal disease) disrupts the active site, whereas the purified R40H mutant has normal catalytic function and this mutation is likely to affect posttranslational processing such as mitochondrial targeting . It appears that most new mutations occur in male sperm and are then passed on to a transmitting heterozygous female . Uncommonly, mild mutations are transmitted by asymptomatic males to their daughters, subsequently resulting in clinical disease of males in future generations . The causes for variable expressivity of these mutations are currently unknown but are likely to involve a combination of environmental and genetic modifiers. Arq Neuropsiquiatr, 1998 Mar, 56(1), 88 - 92 {Survival analysis of acute pyogenic meningitis in children}; Lucena R et al.; In order to determine the survival curves of lethality in acute bacterial meningitis (ABM) in children, we reviewed the charts of all patients admitted to the Hospital Couto Maia from January 1990 to December 1992 . The Kaplan-Meir analysis was used to compare the survival rate and hospitalar permanence of patients with identified pathogens with those whose bacteria were not determined . The same analysis was used to compare the curves of the three most frequent agents . Statistical difference between the identified and nonidentified groups was not observed . The analysis of the three curves shows that the first 24 hours were responsible for the most elevated lethality rate . When the curves are compared, it is clear that S . pneumoniae has the most important intrahospitalar lethality and N . meningitidis the most benign evolution . We conclude that efforts have to be made to determine which variables are related to prognosis of ABM at the first day of hospital admission. J Control Release, 1998 Mar 2, 52(1-2), 109 - 18 Design of microencapsulated chitosan microspheres for colonic drug delivery; Lorenzo-Lamosa ML et al.; Among the different approaches to achieve colon-selective drug delivery, the use of polymers, specifically biodegraded by colonic bacteria, holds great promise . In this work a new system which combines specific biodegradability and pH-dependent release is presented . The system consists of chitosan (CS) microcores entrapped within acrylic microspheres . Sodium diclofenac (SD), used as a model drug, was efficiently entrapped within CS microcores using spray-drying and then microencapsulated into Eudragit L-100 and Eudragit S-100 using an oil-in-oil solvent evaporation method . The size of the CS microcores was small (1.8-2.9 microns) and they were encapsulated within Eudragit microspheres (size between 152 and 233 microns) forming a multireservoir system . Even though CS dissolves very fast in acidic media, at pH 7.4, SD release from CS microcores was delayed, the release rate being adjustable (50% dissolved within 30-120 min) by changing the CS molecular weight (MW) or the type of CS salt . Furthermore, by coating the CS microcores with Eudragit, perfect pH-dependent release profiles were attained . No release was observed at acidic pHs, however, when reaching the Eudragit pH solubility, a continuous release for a variable time (8-12 h) was achieved . A combined mechanism of release is proposed, which considers the dissolution of the Eudragit coating, the swelling of the CS microcores and the dissolution of SD and its further diffusion through the CS gel cores . In addition, infrared (IR) spectra revealed that there was an ionic interaction between the amine groups of CS and the carboxyl groups of Eudragit, which provided the system with a new element for controlling the release . In conclusion, this work presents new approaches for the modification of CS as well as a new system with a great potential for colonic drug delivery. Mutat Res, 1998 May 25, 400(1-2), 169 - 86 DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens; Galloway SM et al.; Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA . These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition . Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved . Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose . Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine {BrdUrd}) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression . Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels . Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control) . Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis . In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment . Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity . Nucleic Acids Res, 1998 Aug 15, 26(16), 3746 - 52 Phosphoesterase domains associated with DNA polymerases of diverse origins; Aravind L et al.; Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases . The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase . The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis . The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity . Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases . The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs . A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization . As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward . The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes . In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members. Pediatrics, 1998 Aug, 102(2 Pt 1), 291 - 5 Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction; Pitkaranta A et al.; OBJECTIVE: To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM) . METHODS: Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA . PATIENTS: Ninety-two children aged 3 months to 7 years during a 1-year period . RESULTS: Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis . HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%) . RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%) . HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%) . Dual viral infections were detected in 5% of children . HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample . Bacterial pathogens were detected in 56 (62%) MEF from 91 children . Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples . No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates . CONCLUSION: These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children. Arch Microbiol, 1998 Sep, 170(3), 201 - 7 Nucleic acid content of synechococcus spp . during growth in continuous light and light/dark cycles Lepp PW, Schmidt TM. Light-limited batch cultures of Synechococcus spp . strains PCC 6301 and WH 8103 exhibited a positive correlation between the specific growth rate and the cellular content of both RNA and DNA . The ratio of RNA to DNA increased with the growth rate in Synechococcus sp . strain WH 8103, as it does in heterotrophic bacteria, but remained constant in Synechococcus sp . strain PCC 6301 . The cellular content of nucleic acids decreased during light periods and increased during dark periods in Synechococcus sp . strain PCC 6301 entrained by 12-h light/ 12-h dark (diurnal) cycles . This result was unexpected in light of experiments demonstrating a circadian increase in transcriptional activity during the subjective light periods and a decrease in transcriptional activity during subjective dark periods . The cyclical variation in cellular nucleic acid content during diurnal cycles appears to be in part a function of the timing of cell division and will influence estimates of in situ growth rates or relative abundances of cyanobacteria using oligonucleotide probes complementary to 16S rRNA. Vet Pathol, 1998 Jul, 35(4), 300 - 3 Subclinical proliferative enteropathy in sentinel rabbits associated with Lawsonia intracellularis; Duhamel GE et al.; Light microscopic and ultrastructural changes of naturally acquired proliferative enteropathy were observed in two of three young sentinel New Zealand White rabbits . The etiologic agent, Lawsonia intracellularis, was demonstrated in the tissues using morphologic, immunohistochemical, and molecular methods . Proliferative enteropathy was associated with infection of villous and crypt enterocytes by intracellular organisms genotypically and antigenically related to L . intracellularis of various other animal species. Proc R Soc Lond B Biol Sci, 1998 Jun 22, 265(1401), 1081 - 90 Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization; Bouchon D et al.; Wolbachia are maternally inherited, intracellular, alpha proteobacteria that infect a wide range of arthropods . They cause three kinds of reproductive alterations in their hosts: cytoplasmic incompatibility, parthenogenesis and feminization . There have been many studies of the distribution of Wolbachia in arthropods, but very few crustacean species are known to be infected . We investigated the prevalence of Wolbachia in 85 species from five crustacean orders . Twenty-two isopod species were found to carry these bacteria . The bacteria were found mainly in terrestrial species, suggesting that Wolbachia came from a continental environment . The evolutionary relationships between these Wolbachia strains were determined by sequencing bacterial genes and by interspecific transfers . All the bacteria associated with isopods belonged to the Wolbachia B group, based on 16S rDNA sequence data . All the terrestrial isopod symbionts in this group except one formed an independent clade . The results of interspecific transfers show evidence of specialization of Wolbachia symbionts to their isopod hosts . They also suggest that host species plays a more important role than bacterial phylogeny in determining the phenotype induced by Wolbachia infection. J Biotechnol, 1998 May 13, 61(3), 175 - 89 A method for motion compensation of a moving nematode Caenorhabditis elegans and its application to frequency analysis of pharyngeal pulsation; Biswas SN et al.; A new sequential image processing method for motion compensation of a moving object with stringy shape has been developed for estimating the pharyngeal pulsation of the nematode Caenorhabditis elegans under several environmental conditions . The method is based on the pixel data transfer on a new image frame while changing the boundary shape and the position but preserving the conformation of the inner structure of an object . All digitized image frames of C . elegans were first converted to motion-compensated images to arrange the pulsation site in the same region of the every transformed frame . The pulsation site was then automatically detected by determining the pixels where the temporal brightness variation was much larger than that of the other pixels . Finally, the pulsation frequency was determined by the Fourier analysis . The validity of our method has been confirmed by analyzing various test data, and the method has been applied for detecting the pharyngeal pulsation frequencies of C . elegans on some environmental conditions, i.e . feed bacteria-free/rich, doping of nerve inactivating ethyl-alcohol and nerve stimulant neurochemical substance of serotonin . The motion compensation method automatically provided reasonable pulsation frequencies which were found to be comparable to those obtained by manual counting . Thus the method is useful for systematic investigations on the variation of pharyngeal pulsation associated with the activity change of the nervous system in environments. Dis Aquat Organ, 1998 Jun 19, 33(2), 93 - 9 Detection of humoral antibodies to Renibacterium salmoninarum in rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar challenged by immersion and in naturally infected populations; Jansson E et al.; Humoral antibodies to heat-stable antigens of Renibacterium salmoninarum (Rs) were detected by enzyme-linked immunosorbent assay (ELISA) in rainbow trout Oncorhynchus mykiss and in Atlantic salmon Salmo salar challenged by immersion . A slow antibody response was found: 3% (1/30) was positive 4 wk after immersion and 72% (26/36) was positive after 8 wk . All 30 fish sampled after 4 wk were found to be infected, as determined by bacterial culture and/or the presence of soluble antigens in the kidney . At 6, 8 and 12 wk after immersion the proportion of positives indicated by ELISA was 58% . The Rs infection was detected by cultivation in 36% of sampled fish collected on the same occasion . Elevated antibody titres to Rs were detected in samples from both Atlantic salmon (59% in 1 farm) and from rainbow trout (20% in 1 of 5 sampled farms) in naturally exposed populations all of which classified positive for bacterial kidney disease (BKD) . Elevated antibody titres were detected among sampled fish from populations of rainbow trout and salmon with clinical BKD . Samples collected from farm populations of rainbow trout, salmon and brown trout Salmo trutta, exposed to Rs but without clinical BKD, were negative in the ELISA, although Rs bacteria or soluble antigens were detected at the same sampling . The antibody ELISA method cannot be recommended for general fish health monitoring purposes, but may be a valuable tool for monitoring the disease progression during controlled experiments. J Bacteriol, 1998 Aug, 180(15), 3853 - 63 Expression of the fixR-nifA operon in Bradyrhizobium japonicum depends on a new response regulator, RegR; Bauer E et al.; Many nitrogen fixation-associated genes in the soybean symbiont Bradyrhizobium japonicum are regulated by the transcriptional activator NifA, whose activity is inhibited by aerobiosis . NifA is encoded in the fixR-nifA operon, which is expressed at a low level under aerobic conditions and induced approximately fivefold under low-oxygen tension . This induction depends on a -24/-12-type promoter (fixRp1) that is recognized by the sigma54 RNA polymerase and activated by NifA . Low-level aerobic expression and part of the anaerobic expression originates from a second promoter (fixRp2) that overlaps with fixRp1 and depends on an upstream DNA region (UAS) located around position -68 (H . Barrios, H . M . Fischer, H . Hennecke, and E . Morett, J . Bacteriol . 177:1760-1765, 1995) . A protein binding to the UAS was previously postulated to act as an activator . This protein has now been purified, and the corresponding gene (regR) has been cloned . On the basis of the predicted amino acid sequence, RegR belongs to the family of response regulators of two-component regulatory systems . We identified upstream of the regR gene an additional gene (regS) encoding a putative sensor kinase . A regR mutant was constructed in which neither a specific UAS-binding activity nor fixRp2-dependent transcript formation and fixR'-'lacZ expression was detected in aerobically grown cells . Anaerobic fixR'-'lacZ expression was also decreased in regR mutants to about 10% of the level observed in the wild type . Similarly, regR mutants showed only about 2% residual nitrogen fixation activity, but unlike nodules induced by nifA mutants, the morphology of those nodules was normal, displaying no signs of necrosis . While regR mutants grew only slightly slower in free-living, aerobic conditions, they displayed a strong growth defect under anaerobic conditions . The phenotypic properties of regS mutants differed only marginally, if at all, from those of the wild type, suggesting the existence of a compensating sensor activity in these strains . The newly identified RegR protein may be regarded as a master regulator in the NifA-dependent network controlling nif and fix gene expression in B . japonicum. Immunol Cell Biol, 1998 Jun, 76(3), 245 - 55 Skin delivery of a hybrid liposome/ISCOM vaccine implicates a role for adjuvants in rapid modulation of inflammatory cells involved in innate immunity before the enhancement of adaptive immune responses; Chin J et al.; There is now compelling evidence that intradermal vaccination with an efficacious adjuvanted antigen triggers a series of coordinated responses characterized initially by the rapid mobilization and recruitment of granulocytes to the lung . Activation of effector cells of the innate immune system is intended to provide surveillance and temporary protective cover at vulnerable mucosal sites while both T and B cell precursors, as well as haematopoietic progenitor cells, are undergoing dramatic reductions in numbers during the first 2-4 days post-vaccination . Some of these events recapitulate those seen after infection with a pathogen . Initial decreases in cell numbers in the thymus and bone marrow (BM) are followed by rapid increases in cellular proliferation in these organs, probably in response to peripheral signals . Vaccine-induced cell death (by apoptosis) in the thymus may provide one of many stimuli needed to up-regulate BM production of progenitor cells, and cells of the B, myeloid and monocytic lineages so that depleted peripheral compartments are replenished . Reconstitution of the latter cell population is critical in ensuring sufficient numbers of APC are generated to deal with extraneous antigen resulting from either vaccination or proliferation of a pathogen . Ultimately, these APC, as effector cells of the innate immune system, must provide pattern recognition of dangerous pathogens and serve to activate appropriate T cell responses . Vaccination not only educates both the innate and adaptive arms of the immune response but also more interestingly, appears to regulate subsequent innate immune responses following exposure to a lethal challenge dose of bacteria . Under these conditions, the rate of loss of BM precursors is greatly attenuated in mice previously vaccinated with adjuvanted antigen compared to unvaccinated controls or mice that had received only antigen . Mice intradermally vaccinated with adjuvanted antigen also displayed increased rates of granulocyte and monocyte recruitment in the lung and spleen . These events occurred very rapidly within 12-36 h of challenge and may be crucial in providing complete protection in vaccinated mice against a challenge dose that was otherwise lethal for unvaccinated controls . Therefore, an important characteristic of an efficacious intradermal vaccine may be the ability to deplete T and B precursors in the thymus and BM lymphoid compartments followed by increased rates of haematopoiesis to re-supply peripheral requirements for granulocytes/monocytes, and T and B cells . Adaptive immunity elicited by intradermal vaccination is, therefore, dependent upon prior activation of the innate immune system. Vaccine, 1998 Jul, 16(11-12), 1087 - 94 Potency testing of a live, genetically attenuated vaccine for salmonids; Marsden MJ et al.; Studies have been performed on the use of a live vaccine for immunization of salmonids against the bacterial disease furunculosis . The protection elicited by a kanamycin-resistant aroA mutant of A . salmonicida (Brivax I) and an unmarked aroA deletion mutant (Brivax II) has been examined, and data compared with protection seen using a freeze-dried Brivax II preparation and a commercial, oil-adjuvanted killed vaccine for furunculosis . Whilst high relative percent survival (RPS) values were seen in fish vaccinated with broth-grown Brivax I after a natural exposure to furunculosis (70-100%), much lower RPS values (30-40%) were seen with Brivax II vaccinated fish after an experimental challenge . Nevertheless, the freeze-dried Brivax II formulation performed as well as the broth-grown Brivax II formulation and a commercial vaccine in these studies . In addition, the environmental impact in terms of bacterial shedding into the tank water has been estimated, and shown to approximately 0.03% of the total inoculum used . Lastly, the freeze-dried formulation has been tested for its ability to infect fish and prime for lymphocyte proliferation and antibody production, relative to broth-grown preparations . In all three experiments no significant differences were seen between fish given the broth-grown and freeze-dried formulations . Such data, together with observations that the freeze-dried live preparation had an extended shelf life with the same potency as freshly grown bacteria, show that the potential exists for a commercially viable live vaccine to be produced for use in aquaculture. Nephrol Dial Transplant, 1998 Jul, 13(7), 1737 - 44 Cytokine production in haemodiafiltration: a multicentre study; Panichi V et al.; BACKGROUND: Bacterial contamination of dialysate may enhance cytokine production in haemodialysis . We tested the hypothesis that intracellular cytokines could be enhanced in a large group of patients exposed to backfiltration of dialysate over a long period of observation . METHODS: The intracellular cytokine (interleukin-1 receptor antagonist and interleukin-1beta) concentrations in chronic uraemic patients undergoing haemodiafiltration, which is known to be associated with backfiltration (Group II, 12 patients), were compared to those found in patients treated with a modified haemodiafiltration modality without backfiltration (Group I, 16 patients), in patients shifted from one modality to the other (Group III, 27 patients) and in 10 patients on haemodialysis (Group IV) in a 1-year multicentre study . Group V comprised 10 healthy volunteers . All dialysis monitors were equipped with dialysate ultrafiltration systems . Dialysate contamination was studied by the LAL and the peripheral mononuclear cell/interleukin-1beta assays in the presence or absence of polymyxin B . RESULTS: Intracellular interleukin-1 receptor antagonist and interleukin-1beta both increased significantly (P < 0.002) but slowly (after 8 months) in Groups II vs I, and during the 4-month period in haemodiafiltration with backfiltration in Group III . The incidence of post/predialysis concentration ratio (over 1.5) increased two- to threefold in patients treated with haemodiafiltration with backfiltration with respect to haemodiafiltration without backfiltration . Results on the assays for LAL (< 0.5 E/ml) and interleukin-1beta (range 80.1-90.2 pg/5 x 10(6) cells; 70.2-81.3 pg/5 x 10(6) cells with polymyxin B) showed a moderate-to-low degree of dialysate contamination . CONCLUSIONS: Backfiltration of dialysate with moderate-to-low degree of contamination may enhance cytokine synthesis in the long term . Thus, the relevance for dialytic strategies aiming at improving dialysate quality and/or at reducing backfiltration is highlighted. Bioorg Med Chem, 1998 Jun, 6(6), 735 - 42 Synthesis and inhibitory activity of novel tri- and tetracyclic quinolines against topoisomerases; Sui Z et al.; A series of isoindolo{2,1-a}- and pyrrolo{1,2-a}quinolines were designed and synthesized for DNA-gyrase and topoisomerase-II inhibition studies . Some of the compounds showed significant activity against the enzymes. Plant J, 1998 Mar, 13(5), 641 - 52 The role of UDP-glucose epimerase in carbohydrate metabolism of Arabidopsis; Dormann P et al.; Uridine 5'-diphospho-glucose-4-epimerase (UDP-Glc epimerase) catalyses the reversible epimerization of UDP-galactose and UDP-glucose . In contrast to bacteria and yeast, expression of the UDP-Glc epimerase gene in Arabidopsis was found not to be induced by galactose . To elucidate the metabolic role of this enzyme, transgenic Arabidopsis plants expressing the respective cDNA in sense or antisense orientation were constructed, leading to a range of plant lines with different UDP-Glc epimerase activities . No alterations in morphology were observed and the relative amounts of different galactose-containing compounds were not affected if the plants were raised on soil . However, on agar plates in the presence of galactose, the growth of different lines was increasingly repressed with decreasing enzyme activity, and an increase in the UDP-Gal content was observed in parallel, whereas the UDP-Glc content was nearly constant . The amount of galactose in the cell wall was increased in plants with low UDP-Glc epimerase activity grown on galactose, whereas the cellulose content in the leaves was not altered . Furthermore, starch determined at different times of the day was highly abundant in plants with low UDP-Glc epimerase activity in the presence of galactose . It is proposed that low endogenous UDP-Glc epimerase activity is responsible for the galactose toxicity of the wild-type . Possible mechanisms by which the starch content might be modulated are discussed. Extremophiles, 1997 Aug, 1(3), 125 - 34 Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus; Choi IG et al.; Aquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95 degrees C . To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5-2 kb genomic DNA fragments of A . pyrophilus . Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identified proteins in the PIR or Genebank databases . These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate . Although the GC ratio of the genome is about 40%, the codon usage of A . pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine . A higher ratio of positively charged amino acids in A . pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability. FEBS Lett, 1998 Jun 23, 430(1-2), 92 - 4 Synthetic human antibodies and a strategy for protein engineering; Winter G; Our understanding of the way antibodies are built in vivo has provided an approach for engineering synthetic human antibodies in bacteria . Such antibodies have not only been raised against foreign antigens, but also against highly conserved antigens or human self-antigens, and have considerable practical potential as reagents for research and also as therapeutics . The approach also has implications for the design of antibody repertoires and for engineering other proteins with desirable binding properties . This review takes a personal view. Curr Eye Res, 1998 Jul, 17(7), 708 - 19 Ion transporters and receptors in cDNA libraries from lens and cornea epithelia; Shepard AR et al.; PURPOSE: To construct tissue-specific cDNA libraries containing copies of genes of ion transporting and receptor proteins in lens and cornea epithelia . To screen the libraries for the cDNA of these molecules and to deduce the protein sequence from the cDNA sequence . METHOD: Lens and corneal cDNA libraries are not commercially available and are difficult to prepare from microdissected single animal eyes or cadaver human eyes due to the small amount of tissue . To overcome these problems, we refined an approach for preparing high efficiency, non-PCR amplified plasmid cDNA libraries, where only nanogram quantities of poly (A) RNA are required . Plasmids were electrotransformed into DH10B bacteria and routinely yielded >10(6) independent colonies with typically >90% recombinants . Randomly selected colonies were subjected to colony PCR analysis to determine the libraries average insert size (typically approximately 1-kb) . Primers to known genes were used in PCR amplification to check for representation of the genes in the cDNA libraries . RESULTS: Using libraries from rabbit cornea epithelium and endothelium, cultured human lens epithelium, and alphaTN4 cells, we have found the libraries to contain the cDNA for 3 common housekeeping proteins expected to be at high copy numbers in all cells . In addition, we identified 22 rare proteins and for many we determined the complete coding regions . These include K+ channels, Cl- channels, Ca++ channels, Na+ channels, three exchangers, the Na+-HCO3- cotransporter, and three kinds of receptors . CONCLUSION: Cornea and lens libraries have been constructed from single cornea or lens preparations . The libraries often contain the cDNA sequences for ion transporting and receptor proteins which are expected to be relatively rare in these cells . Many of these cDNA's have now been sequenced and the encoded proteins identified. Biochem Mol Biol Int, 1998 Jun, 45(2), 349 - 54 Crystals of ribosomal protein L1 from a hyperthermophilic archaeon Methanococcus jannaschii; Tishchenko S et al.; Crystallographic studies of ribosomal proteins from bacteria progressed rapidly during the last decade, though the structures of ribosomal proteins from other kingdoms have not yet been published . Here we describe crystals of archaeal ribosomal protein L1 from Methanococcus jannaschii . The protein crystals were grown in 10% PEG 10 K, 50 mM Hepes-HCl (pH 7.5) in hanging drops equilibrated against 33% PEG 10 K, 100 mM Hepes-HCl (pH 7.5) . The crystals diffract to at least 2.5 A resolution and belong to the space group P1 with cell parameters a = 34.09 A, b = 39.39 A, c = 55.84 A, alpha = 83.65 degrees, beta = 80.38 degrees, gamma = 75.37 degrees. Dis Aquat Organ, 1998 Mar 5, 32(2), 99 - 110 Electron microscope studies of the in vitro phagocytosis of Mycobacterium spp . by rainbow trout Oncorhynchus mykiss head kidney macrophages; Chen SC et al.; The cytological response of rainbow trout Oncorhynchus mykiss head kidney macrophages to ingested Mycobacterium spp . was examined in vitro . Mycobacterium marinum or Mycobacterium sp . TB267 isolated from snakehead fish Channa striata Bloch were opsonised with either fresh rainbow trout serum, serum which had been heat-inactivated, or rainbow trout antiserum against the extracellular products (ECP) of the 2 Mycobacterium spp . A monoclonal antibody against the ECP was also used as an opsonin . Suspensions of macrophages were prepared (1 ml of 1 x 10(7) cells ml-1), mixed with the opsonised bacteria (100 microliters of 2 x 10(9) ml-1), and incubated at 18 degrees C for 0.5, 1, 2, 4 or 6 h to allow phagocytosis to occur . A quantitative evaluation of the phagocytosis of the mycobacteria by the macrophages was carried out by electron microscopy . Macrophage phagosomes and their contents were examined and numbers of intact and partially degraded bacteria determined . Pre-labelling dense granules (secondary lysosomes) with ferritin enabled phagosome lysosome fusion to be identified and their frequency determined . Opsonisation of the mycobacteria was found to greatly enhance the phagocytic and killing activity of the rainbow trout macrophages. FEMS Microbiol Lett, 1998 Jul 1, 164(1), 63 - 8 Regulation of the transcriptional activators AnfA and VnfA by metals and ammonium in Azotobacter vinelandii; Premakumar R et al.; Transcription of the genes encoding molybdenum (Mo)-independent nitrogenases 2 and 3 of Azotobacter vinelandii requires the activators VnfA and AnfA, respectively . The effect of NH4+, Mo, or V (vanadium) was tested on the expression of vnfA-lacZ and anfA-lacZ transcriptional fusions . Mo repressed expression of both fusions whereas NH4+ and V repressed the anfA-lacZ fusion, but not the vnfA-lacZ fusion . Thus the repressive effect on transcription of the anfHDGKOR operon by NH4+, Mo, or V is mediated through their effect on transcription of anfA and the repressive effect of Mo on the vnfHFd and vnfDGK operons is mediated through Mo repression of vnfA transcription . Mo-dependent repression of anfA transcription is influenced but not entirely mediated by the Mo-responsive regulator ModE. Carbohydr Res, 1998 Feb, 307(3-4), 291 - 8 Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing L-iduronic acid and D-QuiNHb4NHb; Hanniffy OM et al.; An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1) . L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose . On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di{(S)-3-hydroxybutyramido}glucose, respectively . This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature. J Dermatol, 1998 Jun, 25(6), 384 - 90 A pediatric case of atypical Mycobacterium avium infection of the skin; Noguchi H et al.; We report a case of cutaneous atypical mycobacteriosis in a 12-year-old healthy girl due to Mycobacterium avium . The cutaneous symptoms were three well-defined subcutaneous nodules on both buttocks and on the posterior surface of the left thigh . One had a fistulous opening on the skin surface . Histopathological examination revealed epithelioid cell granulomas surrounded by dense lymphocytic infiltration and acid-fast bacteria were seen with modified periodic acid-carbol fuchsin staining . Using Ogawa's medium at 37 degrees C, acid-fast bacteria were isolated from the biopsied specimen and identified by the DNA-DNA hybridization method as Mycobacterium avium . In drug susceptibility test, these were resistant to all antituberculous drugs . Oral administration of minocycline 100 mg/day for two months had little effect on the two remaining lesions, which were therefore excised . Based upon reported cases of Mycobacterium avium complex, we considered that our pediatric patient with multiple intradermal or subcutaneous nodules on the buttocks and the thigh exhibited the characteristic symptoms of M . avium infection. Biophys J, 1998 Aug, 75(2), 683 - 94 Model for the light-harvesting complex I (B875) of Rhodobacter sphaeroides; Hu X et al.; The light-harvesting complex I (LH-I) of Rhodobacter sphaeroides has been modeled computationally as a hexadecamer of alphabeta-heterodimers, based on a close homology of the heterodimer to that of light-harvesting complex II (LH-II) of Rhodospirillum molischianum . The resulting LH-I structure yields an electron density projection map that is in agreement with an 8.5-A resolution electron microscopic projection map for the highly homologous LH-I of Rs . rubrum . A complex of the modeled LH-I with the photosynthetic reaction center of the same species has been obtained by a constrained conformational search . This complex and the available structures of LH-II from Rs . molischianum and Rhodopseudomonas acidophila furnish a complete model of the pigment organization in the photosynthetic membrane of purple bacteria. Biochem Biophys Res Commun, 1998 Jul 20, 248(2), 216 - 8 Oligo(dT)-primed synthesis of cDNA by reverse transcriptase in mycobacteria; Rindi L et al.; The possibility that mRNA from Mycobacterium tuberculosis and M . bovis BCG may present polyadenylation at the 3' end was investigated . The total RNA, extracted from the bacterial cells and treated with DNase, was used as substrate for reverse transcriptase (RT)-dependent cDNA synthesis . The RT reaction was primed with oligo(dT) and with downstream specific primers for the genes of the antigens 65 KDa and 85-C . PCR probing of the reaction products for cDNAs of the two mycobacterial genes yielded the expected 225 and 307 bp bands when RT synthesis was primed by oligo(dT) and by downstream specific primers . Reaction products from oligo(dT)-primed RT of RNase-treated RNA and untranscribed RNA, probed by PCR, failed to generate the 225 and 307 bp specific bands . These findings support the existence of polyadenylated tracts in mRNA of mycobacteria that can be targeted by oligo(dT) primers to initiate RT-dependent cDNA synthesis . This may result in an advance in the study of gene expression in these and possibly other bacteria . Biochem Biophys Res Commun, 1998 Jul 9, 248(1), 69 - 74 Epitope mapping of SHP-1 monoclonal antibodies using peptide phage display; Murthy KK et al.; We have characterized the binding epitopes of four monoclonal antibodies for SHP-1, an SH2 domain containing protein tyrosine phosphatase, using two phage displayed random peptide libraries . Three of the antibodies are directed against the phosphatase domain of the molecule and the fourth is toward the NH2-terminal part of the second SH2 domain . The first two antibodies recognize the sequence NANY, amino acid 305 to amino acid 308, numbered in the non haematopoietic form of human SHP-1 sequence . The third antibody binds the sequence P Y W P (amino acids 365 to 368) located toward the middle of the phosphatase domain of the enzyme . The fourth antibody is directed against the first two amino acids, W Y (amino acids 112 and 113), of the second SH2 domain . The specificities of these antibodies are demonstrated by ELISA and western blot using different protein constructs expressed in bacteria . All the antibodies can detect wild type SHP-1, expressed in 293 cells, by western blot analysis, both under denaturing conditions as well as following renaturation . The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitopes are accessible from the outside in the native SHP-1 molecule. Arch Biochem Biophys, 1998 Jul 15, 355(2), 222 - 32 Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences; Kery V et al.; Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway . The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin . Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39 . The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits . Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits . This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria . The active core of CBS forms a dimer of approximately 85 kDa . The dimer is about twice as active as the tetramer . It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet . Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine . We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme . The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies . Biol Reprod, 1998 Jul, 59(1), 69 - 76 Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice; Schumacher JM et al.; Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids . The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids . To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes . We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes . Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette . SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP) . We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP . We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro . A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive . These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP. Berl Munch Tierarztl Wochenschr, 1998 Jun, 111(6), 208 - 10 {Effect of transport stress on the content of endotoxin in blood of slaughter pigs}; Zucker BA et al.; Stress during transportation of slaughter pigs could lead to an increased level of translocation of endotoxin from the gut . In particular, during longtime transportation increased concentrations of endotoxin in blood are likely . Since nonlethal doses of endotoxin could trigger translocation of bacteria from the gut to systemic organs an endogenous contamination of the carcasses also with human pathogens may occur . Therefore, it is necessary to evaluate transportation regimes of slaughter animals also with regard to food-borne infections and intoxications. Infect Immun, 1998 Aug, 66(8), 3964 - 7 Constitutive and inducible green fluorescent protein expression in Bartonella henselae; Lee AK et al.; The green fluorescent protein (GFP) gene was expressed on a plasmid in B . henselae, and GFP-expressing bacteria were visualized by fluorescence microscopy . HEp-2 cells infected with GFP-expressing bacteria were separated from uninfected cells with a fluorescence activated cell sorter . Promoter fusions of B . henselae chromosomal DNA to gfp were examined by flow cytometry, and a B . henselae groEL promoter fusion which induced expression at 37 degreesC was isolated. Infect Immun, 1998 Aug, 66(8), 3892 - 9 Infection of the laboratory mouse with the intracellular pathogen Ehrlichia chaffeensis; Winslow GM et al.; To determine the basis of susceptibility and resistance to human monocytic ehrlichiosis (HME), immunocompetent and immunocompromised mice were infected with Ehrlichia chaffeensis and bacterial loads were measured by PCR and by immunohistochemistry . Immunocompetent (C . B-17 and C57BL/6) mice cleared the bacteria within 10 days, but immunocompromised SCID and SCID/BEIGE mice developed persistent infection in the spleen, liver, peritoneal cavity, brain, lung, and bone marrow and became moribund within 24 days . Both immunocompromised strains lack T and B lymphocytes, but the SCID/BEIGE strain is also deficient in natural killer (NK) cell function . During advanced stages of disease, the infections were associated with wasting, splenomegaly, lymphadenopathy, liver granulomas and necroses, intravascular coagulation, and granulomatous inflammation . Histochemical and immunohistochemical localization studies confirmed the presence of bacteria in tissues, and viable bacteria were cultured from infected animals . The data reveal that T and/or B cells play an essential role during resistance of immunocompetent mice to infection with E . chaffeensis and demonstrate the utility of immunocompromised mice as an experimental model for the study of HME. Infect Immun, 1998 Aug, 66(8), 3643 - 8 An epitope delivery system for use with recombinant mycobacteria; Hetzel C et al.; We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule . This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette-Guerin (BCG) and Mycobacterium vaccae . The broader application of the system was analyzed by preparation of constructs containing peptide epitopes from a range of infectious agents and allergens . We report detailed characterization of the immunogenicity of one such construct, in which an epitope from the Der p1 house dust mite allergen was expressed in M . vaccae . The construct was able to stimulate T-cell hybridomas specific for Der p1, and it induced peptide-specific gamma interferon responses when used to immunize naive mice . This novel expression system demonstrates new possibilities for the use of mycobacteria as vaccine delivery vehicles. Biochemistry, 1998 Jul 21, 37(29), 10363 - 9 The conformational change and active site structure of tetrahydrodipicolinate N-succinyltransferase; Beaman TW et al.; Tetrahydrodipicolinate (THDP) N-succinyltransferase catalyzes the conversion of tetrahydrodipicolinate and succinyl-CoA to L-2-(succinylamino)-6-oxopimelate and CoA . This reaction represents the committed step of the succinylase branch of the diaminopimelate/L-lysine biosynthetic pathway by which many bacteria synthesize meso-diaminopimelate, a component of peptidoglycan, and L-lysine from L-aspartate . The crystal structures of THDP succinyltransferase in complex with the substrate/cofactor pairs L-2-aminopimelate/coenzyme A and L-2-amino-6-oxopimelate/coenzyme A have been determined and refined to 2.0 A resolution . The active site of the enzyme is a long narrow groove located at the interface between two left-handed parallel beta-helix (LbetaH) structural domains of the trimeric enzyme . On binding the amino acid acceptor and cofactor, this groove is covered by residues from the C-terminus of one subunit and a flexible loop excluded from the LbetaH domain of an adjacent subunit to form a tunnel . This conformational change is directly related to interactions between the enzyme and the bound amino acid substrate and cofactor and serves to shield the ligands from bulk solvent and to orient the nucleophilic amino group of the amino acid acceptor toward the mercaptoethylamine group of the cofactor. Mol Cell Biol, 1998 Aug, 18(8), 4744 - 51 Cytoskeletal reorganization by G protein-coupled receptors is dependent on phosphoinositide 3-kinase gamma, a Rac guanosine exchange factor, and Rac; Ma AD et al.; Reorganization of the actin cytoskeleton is an early cellular response to a variety of extracellular signals . Dissection of pathways leading to actin rearrangement has focused largely on those initiated by growth factor receptors or integrins, although stimulation of G protein-coupled receptors also leads to cytoskeletal changes . In transfected Cos-7SH cells, activation of the chemoattractant formyl peptide receptor induces cortical actin polymerization and a decrease in the number of central actin bundles . In this report, we show that cytoskeletal reorganization can be transduced by G protein betagamma heterodimers (Gbetagamma), phosphoinositide 3-kinase gamma (PI3-Kgamma), a guanosine exchange factor (GEF) for Rac, and Rac . Expression of inactive variants of either PI3-Kgamma, the Rac GEF Vav, or Rac blocked the actin rearrangement . Neither wortmannin nor LY294002, pharmacologic inhibitors of PI3-K, could inhibit the actin rearrangement induced by a constitutively active Rac . The inhibition of cytoskeletal reorganization by the dominant negative Vav variants could be rescued by coexpression of a constitutively active form of Rac . In contrast, a Vav variant with its pleckstrin homology (PH) domain missing constitutively induced JNK activation and led to cytoskeletal reorganization, even without stimulation by PI3-Kgamma . This suggests that the PH domain of Vav controls the guanosine exchange activity of Vav, perhaps by a mechanism regulated by D3 phosphoinositides generated by PI3-K . Taken together, these findings delineate a pathway leading from activation of a G protein-coupled receptor to actin reorganization which sequentially involves Gbetagamma, PI3-Kgamma, a Rac GEF, and Rac. Mol Cell Biol, 1998 Aug, 18(8), 4444 - 54 TFII-I regulates Vbeta promoter activity through an initiator element; Cheriyath V et al.; In our effort to understand the transcriptional regulation of naturally occurring TATA-less but initiator (Inr)-containing genes, we have employed the murine T-cell receptor Vbeta 5.2 promoter as a model . Here we show by transient-transfection assays that the Inr binding transcription factor TFII-I is required for efficient expression of the Vbeta promoter in vivo . Mutations in the Inr element that reduced binding of TFII-I also abolished the Vbeta promoter activity by ectopic TFII-I . We further biochemically identified a protease-resistant N-terminal DNA binding fragment of TFII-I, p70 . When ectopically expressed, recombinant p70 bound to the Vbeta Inr element with a specificity similar to that of wild-type TFII-I . More importantly, p70, which lacks independent activation functions, behaved as a dominant negative mutant that inhibited Inr-specific function of wild-type TFII-I . However, the activation functions of p70 were restored when fused to the heterologous activation domain of the yeast activator protein GAL4 . Taken together, these data suggest that TFII-I functions in vivo require an intact Inr element and that the Inr-specific transcriptional functions of TFII-I are solely dictated by its N-terminal DNA binding domain and do not require its own C-terminal activation domain. MMWR Recomm Rep, 1998 Jul 10, 47(RR-10), 1 - 14 Compendium of measures to control Chlamydia psittaci infection among humans (psittacosis) and pet birds (avian chlamydiosis), 1998 . Center for Disease Control and Prevention; Questionnaire survey of proliferative enteropathy on British pig farms; Department of Veterinary Pathology, University of Edinburgh, MidlothianRisk factors for proliferative enteropathy were investigated by means of a postal questionnaire survey of randomly selected British pig farms . Replies were received from 319 (56 per cent) of the 569 questionnaires posted, representing 1.5 per cent of the total number of pig farms in Britain . Thirty-one per cent of the farms had experienced at least one episode of proliferative enteropathy within the previous three years, usually confirmed by their veterinary surgeon . There was a strong association for the occurrence of proliferative enteropathy in herds of over 500 sows (P < 0.005) and in herds with enzootic pneumonia (P < 0.01) . Outbreaks had occurred in five of the six nucleus herds surveyed, the other had only 80 sows . Outbreaks occurred in 32 of 69 herds that had obtained their replacement boars from nucleus herds (P < 0.05), suggesting that infected boars may carry the disease into distant herds . The use of either fully slatted (P < 0.05) or fully meshed floors (P < 0.01) above sunken pits in buildings used to house pigs immediately after weaning, and the use of partially (P < 0.05) or fully slatted floors (P < 0.05) in buildings used to house pigs two to six months old, were risk factors for outbreaks of proliferative enteropathy, compared with the use of straw bedding or solid floors . The destocking of entire buildings containing pigs two to four months old before the introduction of fresh pigs, was associated with a reduced risk (P < 0.05), but the destocking of selected pens rather than the whole building had no such association . The type of diet, or feeding or watering system and the types of buildings used were not identified as risk factors. Antisense Nucleic Acid Drug Dev, 1998 Jun, 8(3), 207 - 14 Interaction of oligodeoxynucleotides with mycobacteria: implications for new therapeutic strategies; Attia SA et al.; The use of synthetic oligonucleotides (ONs) to systematically address new pharmacologic targets in mycobacteria would enhance the introduction of new molecular targets for drug intervention . Oligonucleotides' mechanism of action allows researchers to pursue the importance of particular proteins without the requirement of having purified samples . For this approach to be effective, mycobacteria must be able to transport ONs to their cytoplasm, and if this is not the case, the agents must be otherwise delivered . In this report, we characterize the ability of phosphorothioate (PS) and phosphorodiester (PD) ONs to interact with both Mycobacterium smegmatis and Mycobacterium tuberculosis . In addition, the use of delivery enhancer compounds, ethambutol and PAMAM dendrimer, was evaluated on the ON-mycobacteria interaction . ON interaction was demonstrated to be concentration-dependent, suggesting a possibly active component of the oligonucleotide and bacteria interaction . ON interaction could be increased by the coincubation of the bacteria with the delivery adjuvants . Treatment with ethambutol or dendrimers (fourth generation) was demonstrated to increase ON interaction with both species of mycobacteria although not to the same extent . The results of these preliminary experiments indicate that through use of the proper delivery adjuvant, ON interactions with mycobacteria can be increased . These findings may have implications for probing future antimycobacterial therapeutic targets. Clin Diagn Lab Immunol, 1998 Jul, 5(4), 578 - 82 A euthymic hairless mouse model of Helicobacter pylori colonization and adherence to gastric epithelial cells in vivo; Kimura N et al.; The hairless mouse strain NS:Hr/ICR was examined as a potential small animal model of Helicobacter pylori colonization, adherence to gastric epithelial cells in vivo, and gastritis . Among several small animals tested, NS:Hr/ICR mice proved to be the most highly susceptible to H . pylori infection . Challenge with clinical isolates of H . pylori consisting of either phenotype I or II (VacA and CagA positive and negative, respectively) resulted in colonization by mucus-resident and epithelial cell-adherent bacterial populations . Cell-adherent bacteria resisted 80 cycles of top-speed Vortex washing and were recovered only by homogenization of serially washed glandular stomach tissue, indicating intimate association with the mucosal surface . Immunoperoxidase staining of paraffin sections of gastric tissue from infected mice revealed H . pylori antigens localized in the glandular region of the mucosa, with some colonized areas seen in the vicinity of submucosal mononuclear cell infiltration . The latter inflammatory reaction was observed as a function of the H . pylori phenotype (only type I induced inflammation) and the challenge dose (only those mice challenged with 10(8) CFU or higher showed the reaction) . The NS:Hr/ICR strain of mice is a suitable miniature model of H . pylori infection and may prove useful in the quest for an efficacious mode of treatment for this common infection in humans. Curr Opin Chem Biol, 1998 Apr, 2(2), 182 - 93 Nitrogen cycle enzymology; Ferguson SJ; The minimal nitrogen cycle involves five reduction reactions and three oxidation reactions, each of which poses interesting problems in bioinorganic chemistry, energy transduction and protein structure/function relationships . Many of the major recent developments in this field have depended on the acquisition of protein crystal structures, including structures of enzymes with bound substrates or products and in protein-protein complexes . These enzymes include nitrogenase, nitrite reductases, hydroxylamine oxidoreductase and a fungal nitric oxide reductase. Arch Pediatr Adolesc Med, 1998 Jul, 152(7), 646 - 50 Tuberculosis screening at 2 San Diego high schools with high-risk populations; Pong AL et al.; BACKGROUND: High immigration rates contribute to the high incidence of pediatric tuberculosis (TB) in San Diego, Calif . Adolescents frequently have poor access to health care and may not receive appropriate TB screening . School-based screening has been ineffective in detecting TB in other parts of the country . OBJECTIVE: To determine the prevalence of TB infection and disease in a high-risk population of high school students through school-based screening . DESIGN AND PARTICIPANTS: Cross-sectional study of TB prevalence and an analysis of risk factors for TB infection in students attending 2 San Diego high schools with high percentages of non-US-born students . MAIN OUTCOME MEASURES: Positive induration (> or =10 mm) with Mantoux tuberculin skin test . A chest radiograph or clinical findings consistent with active TB . RESULTS: A total of 744 (36%) students at high school 1 and 860 (57%) students at high school 2 participated . Ninety-five (12.8%) and 207 (24.1%) students, respectively, had positive tuberculin skin test results . One student had a chest radiograph that showed active TB . Smear for acid-fast bacteria and culture for Mycobacterium tuberculosis had negative results . Vietnamese, Filipino, and Latino ethnic groups were significantly more likely to have positive tuberculin skin test results than the white population (P<.05) . Non-US-born students were significantly more likely to have positive tuberculin skin test results than US-born students in all ethnic groups except the Latino group . CONCLUSION: Although treatment of TB coupled with aggressive public health investigation is the most cost-beneficial way of preventing TB, targeted school-based screening may be an effective way of detecting TB infection in high-risk populations with poor access to health care. Gen Dent, 1998 Jan-Feb, 46(1), 34 - 8, 40 Chlorhexidine, fluoride varnish, and xylitol chewing gum: underutilized preventive therapies? Anusavice KJ. The successful implementation of a preventive dentistry program depends, to a large extent, on the compliance of the patient . The scheduled program would include: recall appointments, all instructions relative to oral hygiene, use of nightly fluoride rinses, and control of diet . To ensure that high-risk patients who have cariogenic bacteria are adequately treated, chlorhexidine rinses may be required on a periodic basis . The patient's level of risk must determine all treatment decisions . For low-risk patients, the times between recall appointments can be extended when evidence of caries arrest and remineralization can be documented . High-risk patients should be recalled at least every three months, until evidence of lesion arrest and/or remineralization has been documented . For patients with extremely low saliva flow rates, the combined chlorhexidine and fluoride method may be required . If the caries risk is still judged to be high according to bacteria counts and/or evidence of further lesion development or progression, more frequent applications of chlorhexidine may be required . Because fluoride varnish is generally more effective on smooth surfaces than on fissure sites, moderate caries-risk patients should receive fluoride varnish on smooth surfaces, and sealants, when indicated, on fissure sites . As the caries risk of the patient is reduced to a low-risk level, less frequent use of fluoride-containing or fluoride releasing products is indicated, and there can be longer periods between recall examinations . Three applications of fluoride varnish, applied to a single week, appear to provide greater caries protection than two applications per year . Attempts should be made to ensure that the varnish is applied immediately after cleaning the teeth and protected as long as possible after the varnish has been applied (preferably at least 10 hours) . Fluoride varnish appears to be as effective as topical fluoride gel and may be safer . Thus, a greater frequently of application is permitted without a significant risk of fluorosis . Prevention is more cost effective as the patient shifts from a high-risk level to a low-risk level . Recall appointments can subsequently be extended and more conservative prevention treatments are warranted . Over an extended treatment period, the cost for the preservative dentistry option should be comparable to and perhaps less than the cost of placing and replacing dental restorations. Tuber Lung Dis, 1997, 78(1), 67 - 73 Expression of memory immunity in the lung following re-exposure to Mycobacterium tuberculosis; Cooper AM et al.; OBJECTIVE: To examine the memory immunity expressed in the lung in response to a low-dose aerosol challenge . DESIGN: Memory-immune C57BL/6 mice were generated by infection followed by drug treatment with isoniazid and rifabutin . Both memory-immune and naive mice were then rechallenged via both the aerosol and intravenous routes . The growth of bacteria in target organs, the expression of cytokines within these organs and the ability of T cells to recognize selected mycobacterial protein antigens were determined over time . RESULTS: There was a finite delay before immunity was expressed in the lungs of the memory-immune mice . This was in contrast to the immediate control of bacterial growth seen in the liver of intravenously challenged mice . In both cases, the expression of interferon-gamma (IFN-gamma) mRNA in the target organ correlated with the control of bacterial growth . Memory immunity in the spleen and lung differed: whereas splenic T cells strongly recognized the major Ag85 protein, the 45 kDa protein, and a synthetic peptide representing the ESAT molecule, only the Ag85 molecule was recognized by T cells harvested from thoracic lymph nodes after pulmonary rechallenge . CONCLUSIONS: Immunity, as mediated by IFN-gamma, is expressed more slowly following an aerosol rechallenge and appears to be restricted in terms of antigen specificity . Moreover, very strong levels of memory immunity can prevent progressive disease in the lungs, but cannot prevent the establishment of secondary infection. Anal Chem, 1998 Jul 1, 70(13), 2731 - 6 Combining MALDI mass spectrometry and biomolecular interaction analysis using a biomolecular interaction analysis instrument; Sonksen CP et al.; Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been combined with biomolecular interaction analysis (BIA) in a Biacore instrument . A method has been developed for the recovery of the affinity-bound molecules from the sensor chip in a few microliters ready for mass spectrometr