Microorganism

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 spectrometric analysis . The procedure is illustrated with two molecular systems which exemplify antibody-antigen and DNA-protein interactions . In both cases, femtomole quantities of the affinity-bound proteins were eluted and subsequently detected by MALDI-MS . Whereas the Biacore analysis yields the surface concentration of protein bound to the sensor chip, identity of the bound compounds is revealed in the second step by accurate molecular mass determination . Combining the information of the two analyses allows calculation of the total surface molar concentration of affinity-bound molecules.

Nippon Yakurigaku Zasshi, 1998 May, 111(5), 289 - 96
{Rodent models of Helicobacter pylori infection and their utility}; Takahashi S et al.; Small animal models for Helicobacter pylori (H . pylori) infection have been widely examined and developed . Recently, the novel laboratory strain (the Sydney strain) of H . pylori, having a high ability to colonize the gastric mucosa of normal mice, was established . In the mice infected with the Sydney strain, chronic gastritis slowly develops, progressing to severe atrophy . The Sydney strain may become the standard one for experimental research . On the other hand, mice expressing Leb antigen were genetically produced . H . pylori well colonizes the gastric mucosa of the transgenic mice, but the gastric pathology remains unclear . However, formation of gastric ulcers has not been observed in mice . In contrast, H . pylori chronically infects Mongolian gerbils and causes severe gastritis and gastric ulcers . The H . pylori-induced gastric ulcers do not heal spontaneously, but are cured by drugs . To examine H . pylori colonization in the gastric mucosa and eradication of the bacteria, mouse models are satisfactory . However, in the case of studies on H . pylori-associated gastric pathology, the Mongolian gerbil model is better . Although these models have both merits and demerits, they are useful for elucidation of the pathogenesis of H . pylori-associated diseases and for development of drugs and therapies.

Clin Diagn Lab Immunol, 1998 Jul, 5(4), 452 - 5
Intrathecal synthesis of immunoglobulins in eosinophilic meningoencephalitis due to Angiostrongylus cantonensis; Dorta-Contreras AJ et al.; Eosinophilic meningoencephalitis due to the nematode Angiostrongylus cantonensis, which is endemic to Cuba, occurs in children and is due to accidental contact with soil snails . The course is less often fatal than in adult patients in southeastern Asia . Cerebrospinal fluid (CSF) and serum samples from 24 pediatric patients were analyzed and evaluated in CSF/serum quotient diagrams (Reiber graphs) to characterize the neuroimmunological response and the blood-CSF barrier dysfunction that occur in the course of the disease . At the time of the first diagnostic lumbar puncture, together with eosinophilic pleocytosis (1,920 +/- 400 cells/microl), intermediate blood-CSF barrier dysfunction (i.e., an increased CSF/serum albumin quotient) with no intrathecal immunoglobulin G (IgG), IgA, and IgM class response was observed in all cases . Seven days later, at the time of early clinical recovery, the blood-CSF barrier dysfunction was normalized in 75% of the patients, but meanwhile, intrathecal immunoglobulin synthesis emerged in all cases, as either a two-class response (IgG and IgA in 85% of the patients) or a three-class response (IgG, IgA, and IgM; 30%) . The fraction of eosinophilic cells (40%) remained large despite a decreasing total cell count . The neuroimmunological pattern of this inflammatory response to the parasite and its toxins is discussed with regard to the CSF patterns of other infectious diseases caused by bacteria or viruses.

Biochemistry, 1998 Jul 14, 37(28), 10006 - 15
Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis; Hu Q et al.; We have investigated the spectroscopic properties of two classes of light-harvesting 2 (LH2, B800-850) mutants of Rhodobacter capsulatus obtained by combinatorial mutagenesis to the C-terminal half of the beta-apoprotein: a pseudoLH2 (pLH2) class, in which the 800-nm absorption was normal but the 850-nm peak was blue-shifted by up to 14 nm, and the other a pseudoLH1 (pLH1) class, which lacked the 800-nm absorption band and showed 850-nm absorption red-shifts of up to 30 nm . In several of the pLH1 antennae, carotenoid depletion contributed to the phenotype, while in the pLH2 complexes there was some carotenoid enrichment . A number of mutants from each class have also been characterized by low-temperature absorption and fluorescence spectroscopy, resonance Raman spectroscopy, and circular dichroism . In all of the mutants investigated, the B850 bacteriochlorophyll a binding site remained intact, conserving both the hydrogen bonding environment of the chromophores and their conformation and liganding . In contrast, the intensity of the CD spectra of pLH1 complexes was considerably reduced, relative to that of wild-type or pLH2 complexes, consistent with alterations in the interactions between pigments and in their relative orientation . Elevated fluorescence polarization over the red wing of the B850 band in the pLH2 complexes indicated a reduction of exciton mobility within the ring of BChl molecules . Possible structural alterations governing the spectral properties of the different mutants are discussed.

Alcohol, 1998 Aug, 16(2), 167 - 75
Role of PKC and tyrosine kinase in ethanol-mediated inhibition of LPS-inducible nitric oxide synthase; Greenberg SS et al.; Ethanol increases human and animal susceptibility to opportunistic lung infections in part by suppression of endotoxin (LPS) and bacteria-mediated upregulation of inducible nitric oxide synthase (iNOS) in alveolar macrophages (AM) . LPS and cytokine-induced NOS mRNA are dependent on NF-kappaB/Rel (NFkappaB) and Activator Protein-1 (AP-1), which are regulated in turn by protein kinase C and tyrosine kinase-dependent phosphorylation . ETOH does not directly inhibit NFkappaB or AP-1, in vivo, but rather inhibits LPS-induced activation of the MEKK/MAP kinase system and inhibition of inhibitory protein IkappaBalpha required for formation of AP-1 and NFkappaB, respectively . in AM . Both transcription factors are involved iNOS mRNA transcription . LPS-induced upregulation of MEKK/MAP tyrosine kinase upregulates NADPH oxidase activity and oxygen free radical formation required for activation of NFkappaB and AP-1 and phosphorylation of IkappaBalpha . LPS downregulates endogenous calcium-sensitive PKC isozymes (PKCdelta), which repress iNOS mRNA expression . ETOH inhibits LPS-induced upregulation of iNOS mRNA by preventing its ability to decrease PKCdelta and upregulate tyrosine kinase-mediated phosphorylation . This effect of ETOH is prevented by inhibitors of PKC and tyrosine kinase . The data support the hypothesis that ETOH inhibits LPS-induced upregulation of iNOS mRNA by interfering with the phosphorylation processes involved in activation of the nuclear transcription factors NFkappaB and AP-1.

Nat Struct Biol, 1998 Jul, 5(7), 602 - 11
A protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus contains two thioredoxin fold units; Ren B et al.; Protein disulfide bond formation is a rate limiting step in protein folding and is catalyzed by enzymes belonging to the protein disulfide oxidoreductase superfamily, including protein disulfide isomerase (PDI) in eucarya and DsbA in bacteria . The first high resolution X-ray crystal structure of a protein disulfide oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus reveals structural details that suggest a relation to eukaryotic PDI . The protein consists of two homologous structural units with low sequence identity . Each unit contains a thioredoxin fold with a distinct CXXC active site motif . The accessibilities of both active sites are rather different as are, very likely, their redox properties . The protein shows the ability to catalyze the oxidation of dithiols as well as the reduction of disulfide bridges.

Nat Struct Biol, 1998 Jul, 5(7), 568 - 70
Structural and dynamic changes of photoactive yellow protein during its photocycle in solution; Rubinstenn G et al.; Light irradiation of photoactive yellow protein (PYP) induces a photocycle, in which red-shifted (pR) and blue-shifted (pB) intermediates have been characterized . An NMR study of the long-lived pB intermediate now reveals that it exhibits a large degree of disorder and exists as a family of multiple conformers that exchange on a millisecond time scale . This shows that the behavior of PYP in solution is different from what has been observed in the crystalline state . Furthermore, differential refolding to ground state pG is observed, whereby the central beta-sheet and parts of the helical structure are formed first and the region around the chromophore at a later stage.

Mol Cell Probes, 1998 Jun, 12(3), 175 - 80
Detection of adenovirus in the waters of the Seine River estuary by nested-PCR; Castignolles N et al.; Several systems for isolating viruses from environmental samples have been tested . The most promising method is based on genomic amplification . The authors attempted to detect adenovirus in nucleic-acid extracts from the Seine River estuary by a two-step amplification of a 220-bp segment of the conserved coding region of type 2 adenovirus hexon protein L3 . The primers used in this study detected the most prevalent adenovirus serotypes in human disease in France, but not other virus strains or bacteria . The sensitivity of the nested polymerase chain reaction (PCR) amplification was estimated to be 10(2) copies of the adenovirus target sequence per ml of Seine River water . Nucleic-acid extracts from Seine River estuary waters were analysed and some tested positive for the presence of adenoviruses.

Curr Opin Biotechnol, 1998 Apr 1, 9(2), 157 - 63
Refolding of recombinant proteins
Clark EDB.
Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion body protein can be successfully refolded . Aggregation is the leading cause of decreased refolding yields . Developments during the past year have advanced our understanding of the mechanism of aggregation in in vitro protein folding . New additives to prevent aggregation have been added to a growing list . A wealth of literature on the role of chaperones and foldases in in vivo protein folding has triggered the development of new additives and processes that mimic chaperone activity in vitro.

J Oral Sci, 1998 Mar, 40(1), 31 - 6
Rapid diagnosis of oral tuberculosis by amplification of Mycobacterium DNA from paraffin embedded specimens; Komiyama K et al.; The polymerase chain reaction (PCR) assay is a powerful tool for quick diagnosis of various infectious diseases . We applied this technique as well as conventional histopathological examination to diagnose oral tuberculosis . Ziehl-Neelsen staining of oral mucosal specimens often fails to detect Mycobacterium (M.) tuberculosis due to the low number of bacteria in the tissue . Specific primers and probes were synthesized based upon the nucleotide sequence of the 65 kDa membrane protein of M . tuberculosis . DNA extracted from the paraffin-embedded tissue was amplified using taq polymerase . PCR assay detected M . tuberculosis in 5 of 6 samples . Although the gene segments from these species were quite similar, the gamma 32P labeled noligonucleotide probes distinguished between M . tuberculosis and M . fotuitum by southern blot hybridization . In all specimens that were Ziehl-Neelsen negative, M . tuberculosis DNA was detected by PCR . These results suggest that PCR is a useful means of diagnosing mycobacterium infection.

Aliment Pharmacol Ther, 1997 Dec, 11(6), 1115 - 8
Comparison of two 1-week low-dose omeprazole triple therapies--optimal treatment for Helicobacter pylori infection?
Goh KL, Parasakthi N, Chuah SY, Cheah PL, Lo YL, Chin SC.
OBJECTIVES: To determine and compare the efficacy and tolerability of two 1-week regimen comprising omeprazole, clarithromycin and amoxycillin or metronidazole in the eradication of Helicobacter pylori, and to determine the influence of bacterial resistance to metronidazole and clarithromycin on the outcome of treatment . PATIENTS AND METHODS: Patients with unequivocal evidence of H . pylori infection based on culture, histology and rapid urease test of both antrum and corpus biopsies were recruited for the study . The study was a randomized, investigator-blind, comparative study . Patients received either omeprazole 20 mg o.m., clarithromycin 250 mg b.d . and amoxycillin 500 mg b.d . (OAC) or omeprazole 20 mg o.m., metronidazole 400 mg b.d . and clarithromycin 250 mg b.d . (OMC) for 1 week . Patients were assessed for successful eradication, which was defined as absence of bacteria in all tests (culture, histology and urease test on both antral and corpus biopsies), at least 4 weeks after completion of therapy . RESULTS: Eighty-two patients were recruited for the study . Eradication rates on intention-to-treat analysis were--OAC: 36/41 (87.8%, 95% CI: 73.8, 95.9); OMC: 33/41 (80.5%, 95% CI: 65.1, 91.2) . On per protocol analysis were--OAC: 36/40 (90%, 95% CI: 76.3, 97.2); OMC: 32/38 (84.2%, 95% CI: 68.7, 94.0) . All side-effects encountered were mild and no patient discontinued treatment because of intolerance to medications . The most common side-effects were altered taste (OAC 31.7%, OMC 53.7%) and lethargy (OAC 14.6%, OMC 19.5%) . Pre-treatment metronidazole resistance was encountered in 34/63 (54.0%) patients . No bacterial strains were found with primary resistance to clarithromycin . Metronidazole resistance did not significantly affect eradication rates . Emergence of resistance to clarithromycin was not seen post-therapy . CONCLUSIONS: Both the OAC and the OMC regimens were convenient and well-tolerated treatments for H . pylori . However, eradication rates were lower than anticipated.

Biomaterials, 1998 Apr-May, 19(7-9), 683 - 90
A histologic evaluation of eight cases of failed dental implants: is bone overheating the most probable cause?
Piattelli A, Piattelli M, Mangano C, Scarano A.
Biomechanical overloading has been stated to be, overall, the major cause of implant failures . Very important can be, however, in the etiopathogenesis of early implant failure, the overheating of the surgical site . The authors present eight cases of implant loss most probably due to bone overheating, even if other causes cannot be excluded . The microscopical picture, in all cases, was composed by the same features: (1) presence of bone sequestra; (2) no regeneration of the peri-implant bone; (3) presence of an inflammatory infiltrate in the gap between bone and implant; (4) no organization of the peri-implant bone clot; (5) presence of a compact and mature bone around the implant; (6) presence of bacteria around the implant and the necrotic bone.

Biomaterials, 1998 Apr-May, 19(7-9), 643 - 9
Microscopical features in retrieved human Branemark implants: a report of 19 cases; Piattelli A et al.; The authors present a histologic analysis of 19 Branemark titanium implants retrieved for different causes: four implants were removed for abutment fracture, one for dental nerve dysesthesia, two for bone overheating, two for peri-implantitis, nine for mobility, one for unknown causes . In the implants removed for fracture a high bone-implant contact percentage was present (71.83 +/- 4.96%) with compact, mature bone at the interface . The picture of the failure due to bone overheating was characteristic with the presence of bone sequestra and of a gap between implant and bone filled by lymphocytes and plasma cells: many bacteria surrounded the necrotic bone and no newly regenerated bone was present . In peri-implantitis an inflammatory infiltrate was observed in the peri-implant tissues: a dense fibrous connective tissue was present around implants failed for mobility . The microscopical picture is certainly extremely important in identifying the causal determinants of an implant failure.

J Dent Res, 1998 Jul, 77(7), 1515 - 9
Submandibular salivary proteases: lack of a role in anti-HIV activity; Kennedy S et al.; Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity . However, less information is available regarding proteases produced by salivary glands and present in salivary secretions . In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities . These have been characterized in terms of their susceptibility to a series of protease inhibitors . The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors . We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva . This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid . Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.

Cell Tissue Res, 1998 Aug, 293(2), 327 - 36
Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata); Westermann B et al.; The foregut, stomach, caecum, midgut, and rectum of the digestive tract of Nautilus pompilius L.were investigated with ultrastructural and enzyme-cytological methods . Three different cell types were identified within the lamina epithelialis mucosae: main cells, goblet cells, and cells with secretory granules . The main cell type is the epithelial cell with microvilli, a basal nucleus surrounded by dictyosomes, rough endoplasmic reticulum, mitochondria, and electron-dense granules identified as lysosomes in the apical part of the cell . In the caecum this cell type contains endosymbiotic bacteria . The presence of endocytotic vesicles and the storage of lipids in the caecum indicate that this organ is involved in the process of absorption . In the caecum and the longitudinal groove of the rectum the main cells are, in addition, ciliated, facilitating the transport of food particles and faeces . Two types of goblet cells are found in all organs except in the stomach, forming a gliding path for food particles and protecting the epithelium . In the foregut and rectum, cells with electron-dense granules were recognized as the third type . The conspicuous secretory cells of the rectum represent a delimited rectal gland; its possible biological function is discussed . The tunica muscularis in all organs of the digestive tract consists of obliquely striated muscle cells innervated by axons containing transparent, osmiophilic and dense-cored vesicles . Positive reactions for acid and alkaline phosphatase, monoamine oxidase, beta-glucuronidase, and trypsin- and chymotrypsin-like enzymes are localized in the lamina epithelialis mucosae.

Blood Coagul Fibrinolysis, 1998 Apr, 9 Suppl 2, S25 - 37
Antithrombin prevents endotoxin-induced pulmonary vascular injury by inhibiting leukocyte activation; Okajima K; Replacement of antithrombin has proved to be effective for treating disseminated intravascular coagulation . The administration of antithrombin is also useful for preventing organ failure in animals challenged with endotoxin or bacteria, and it increases the survival rate of such animals . Since inhibition of coagulation abnormalities by heparin failed to prevent organ failure in animals challenged with bacteria, antithrombin might exert therapeutic effects independently of its anticoagulant effect . These therapeutic mechanisms of antithrombin have been explored by using animal models of septicemia . Antithrombin prevents pulmonary vascular injury by inhibiting leukocyte activation in rats challenged with endotoxin . A higher dose of antithrombin was required to prevent pulmonary vascular injury than was required to inhibit disseminated intravascular coagulation . This preventive effect of antithrombin is mediated by the promotion of endothelial release of prostacyclin, an inhibitor of leukocyte activation . An interaction between antithrombin and heparin-like glycosaminoglycans on the endothelial cell surface appears to be important for this effect . Heparin inhibits such therapeutic effects of antithrombin by preventing it from interacting with the cell surface heparin-like glycosaminoglycans . Since activated leukocytes are of critical importance in patients with sepsis-associated organ failure, this anti-inflammatory activity of antithrombin may explain why it can prevent organ failure as well as coagulation abnormalities in patients with sepsis.

Nat Biotechnol, 1998 Jul, 16(7), 652 - 6
Man-made cell-like compartments for molecular evolution; Tawfik DS et al.; Cellular compartmentalization is vital for the evolution of all living organisms . Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype . We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions . These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts . A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product . In other compartments, substrates attached to genes that do not encode catalysts remain unmodified . Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product . We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system . A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion . Genes encoding HaeIII methyltransferase were selected from a 10(7)-fold excess of genes encoding another enzyme.

Mol Cell, 1998 May, 1(6), 917 - 24
Activator-mediated recruitment of the RNA polymerase II machinery is the predominant mechanism for transcriptional activation in yeast; Keaveney M et al.; Eukaryotic transcriptional activators bind to enhancer elements and stimulate the RNA polymerase II (pol II) machinery via functionally autonomous activation domains . In yeast cells, the normal requirement for an activation domain can be bypassed by artificially connecting an enhancer-bound protein to a component of the pol II machinery . This observation suggests, but does not necessarily indicate, that the physiological role of activation domains is to recruit the pol II apparatus to promoters . Here, we show that transcriptional stimulation does not occur when the activation domain is physically disconnected from the enhancer-bound protein and transferred to components of the pol II machinery . The observation that autonomous activation domains are functional when connected to enhancer-bound proteins but not to components of the pol II machinery strongly argues that recruitment is the predominant mechanism for transcriptional activation in yeast.

Science, 1998 Jul 10, 281(5374), 207 - 10
Acquisition and utilization of transition metal ions by marine organisms; Butler A; Recent research has revealed that trace metals, particularly transition metals, play important roles in marine productivity . Most of the work has been on iron, which shows a nutrient-depleted profile in the upper ocean . Marine organisms have a variety of means for acquiring iron and other transition metal ions that differ from those of terrestrial organisms.

Parasitol Res, 1998 Jun, 84(6), 476 - 7
Scanning electron microscopy of Blastocystis hominis cysts; Zaman V et al.; Scanning electron microscopy of Blastocystis hominis cysts reveals that some cysts have an outer coat, whereas others are naked . If intact, the outer coat forms a fan-like structure around the cyst and its surface is granular . The fragmented outer coat adheres to other cysts and bacteria, forming irregular clumps.

Carcinogenesis, 1998 May, 19(5), 755 - 64
Sequence specific mutations induced by N-nitrosodimethylamine at two marker loci in metabolically competent human lymphoblastoid cells; Dobo KL et al.; N-Nitrosodimethylamine (NDMA) is a potent mutagen and animal carcinogen to which many people are exposed through the consumption of contaminated food and the use of tobacco products . Although the mutational specificity of NDMA has been studied in bacteria, little is known about the specific types of mutations induced by NDMA in the human genome . Knowledge of the mutational spectrum of NDMA in human genes may help to substantiate the role of NDMA in the etiology of human cancers . In the current study, the mutational spectrum of NDMA was characterized at the tk and hprt loci, in human lymphoblastoid cells capable of metabolically activating NDMA . A number of patterns were observed among NDMA-induced mutations . At both marker loci, G:C-->A:T transitions dominated the mutational spectrum of NDMA, which were indicative of the mutagenicity of the O6meG lesion . In addition, the majority of G:C-->A:T mutations occurred at guanines 3' to another guanine . Almost all of these mutations originated on the non-transcribed strand, which suggests that transcription-coupled repair influenced the distribution of G:C-->A:T transitions at the tk and hprt loci . Furthermore, the observation of hotspots for G:C-->A:T mutations, within both loci, suggests that differential repair kinetics may exist, and consequently affect the distribution of mutations . Finally, a comparison of the site specificity of G:C-->A:T mutations at the tk and hprt loci, indicated that the gene used for mutational analysis influenced the site specificity of NDMA-induced mutations, and possibly reflects the number of 5'-GG-3' sites in the tk and hprt loci that when mutated would yield a mutant phenotype.

Crit Care Med, 1998 Jun, 26(6), 1020 - 4
Septic shock: an analysis of outcomes for patients with onset on hospital wards versus intensive care units; Lundberg JS et al.; OBJECTIVE: To determine if early interventions for septic shock were associated with reduced mortality . DESIGN: Retrospective cohort study . SETTING: University hospital intensive care unit (ICU) and general wards . PATIENTS: Forty-one consecutive patients prospectively identified with positive blood cultures and septic shock . Although all patients were eventually treated in an ICU, ten (24%) patients were on a general ward at the onset of septic shock, and 31 (76%) were in an ICU setting . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: Over a period of 9 mos, a cohort of 41 patients who had positive blood cultures and septic shock was prospectively identified . The 28-day crude mortality was 46% (19 deaths) . We compared the management of septic shock and outcome for patients on a general ward vs . those patients in an ICU setting . Of the ten patients on the ward at time of shock onset (median age 55.5 yrs; median Acute Physiology and Chronic Health Evaluation {APACHE} II score of 18.5), seven (70%) died . In contrast, the 31 patients receiving intensive care when shock developed were older and more ill (median age 66 yrs; median APACHE II 24), yet had a mortality of 39% (12 deaths) . The odds ratio (OR) for death for ward patients compared with ICU patients was 3.57 (p=.17) . In a multivariate logistic regression analysis, two risk factors for mortality were important: APACHE II score (p=.015) and ward status (p=.08) . Candida species in the bloodstream is known to have a high attributable mortality . When type of bloodstream pathogen (Candida species vs . bacteria) was added to the model, APACHE II (OR 2.64 for 10-unit increase) remained significant (p=.014), but ward status (OR 3.97) became statistically nonsignificant (p=.222) . The patients who were on a general ward when their shock developed had a median delay of 67 mins before transfer to an ICU setting . Ward patients received an intravenous fluid bolus after a median delay of 27 mins, whereas those in the ICU who received a fluid bolus did so after a median of 15 mins (p=.48) . Ward patients also had a median delay of 310 mins to receive inotropic support compared with a median 22.5 mins (p=.037) for the patients in an ICU setting when shock started . CONCLUSIONS: The data suggest that for patients with septic shock on wards, there were clinically important delays in transfer of patients to the ICU, receipt of intravenous fluid boluses, and receipt of inotropic agents . However, the most powerful predictors of mortality were APACHE II scores and bloodstream infection with Candida species.

Poult Sci, 1998 Jul, 77(7), 1036 - 44
Characterization of turkey spermiophages with regard to traits common to macrophages; Korn N et al.; Turkey peritoneal exudate cells (PEC) and spermiophages (SMO) were assayed for characteristics of macrophages . The PEC elicited by i.p . injection of 3% Sephadex and SMO isolated from semen using Percoll were cultured in Dulbecco's Modified Eagle Medium supplemented with 20% bovine calf serum (DMEM-20) for 24 h at 41 C in 5% CO2 to provide adherent cells for assays . Most PEC and SMO showed esterase activity (99.3 +/- 0.6 and 98.8 +/- 0.9%, respectively), and exhibited nonspecific phagocytosis of carbon (89.5 +/- 3.6 and 95.3 +/- 0.6%, respectively), zymosan (26.5 +/- 7.6 and 24.3 +/- 2.5%, respectively), bacteria (11.3 +/- 0.8 and 9.3 +/- 0.3%, respectively), and opsonized and nonopsonized SRBC . Maximum uptake of SRBC was seen by 2 h for PEC but not until 4 h for SMO . At time of maximum uptake, SRBC were noted in 35 to 40% of PEC but only in 15 to 20% of SMO . Turkey IgG-FITC bound to both PEC and SMO, but goat anti-turkey IgG-FITC bound only to SMO . Increased nitrite was found in turkey semen after 24 h storage, with highest levels in samples in which SMO were added . Nitrite production was demonstrated using adhered PEC, but SMO could not be tested due to low cell numbers . This research clearly identifies SMO as having macrophage-like activities . Accordingly, these cells may possess the ability to process and present antigen via histocompatibility receptors . Such activity could lead to immune directed responses, including antibody production or activation of cytotoxic T-lymphocytes.

Mol Biochem Parasitol, 1998 May 1, 92(2), 275 - 89
Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae; Loukas A et al.; Cysteine proteases play vital biological roles in both intracellular and extracellular environments . A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F . TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC . Excretory/secretory (TES) products of T . canis larvae did not cleave either substrate . Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T . canis larvae was then obtained from an expressed sequence tag (EST) project . The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases . Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity . Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds . The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX . Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not . Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

Immunol Lett, 1998 Apr, 61(2-3), 119 - 25
Anti-Lewis X antibody and Lewis X-anti-Lewis X immune complexes in Helicobacter pylori infection; Chmiela M et al.; A molecular similarity of Lewis antigens expressed by Helicobacter pylori bacteria and those present in human gastric mucosa has been recognised as a cause of autoimmunity involved in the pathogenesis of chronic type B gastritis and gastric and duodenal ulcers . In this study, the expression of Lewis X determinants was found on 56% of H . pylori strains isolated from patients with chronic gastritis/gastroduodenitis . Anti-Lewis X IgG as well as Lewis X-anti-Lewis X IgG complexes were detected in the sera from patients and even more frequently in the sera from healthy blood donors producing antibodies against surface antigens of H . pylori . It suggested that the initial H . pylori-induced lesions were independent of anti-Lewis X antibody production . When H . pylori bacteria expressing Lewis X antigen were treated with anti-Lewis X monoclonal antibody (mAb) of IgM isotype, they were more susceptible to ingestion by polymorphonuclear leukocytes (PMN) than untreated bacteria . This fact may lead us to believe that anti-Lewis X antibody limits the growth of H . pylori on gastric mucosa.

Inflamm Res, 1998 May, 47(5), 201 - 10
The inflammatory basis of trauma/shock-associated multiple organ failure; Yao YM et al.; Multiple alterations in inflammatory and immunologic function have been demonstrated in clinical and experimental situations after trauma and hemorrhage, in particular the activation of various humoral (e.g . complement, coagulation) and cellular systems (neutrophils, endothelial cells, macrophages) . As a consequence of this activation process there is synthesis, expression and release of numerous mediators (toxic oxygen species, proteolytic enzymes, adherence molecules, cytokines), which may produce a generalized inflammation and tissue damage in the body . Mediators are responsible for ongoing interactions of different cell types and for amplification effects through their networks and feedback cycles, finally leading to a sustained inflammation and multiple organ damage in the body . In the setting of trauma/shock, many activators including bacterial as well as non-bacterial factors may be present that will induce local and systemic inflammatory responses . Although the potential role of bacteria/endotoxin translocation and its clinical relevance remains controversial, many lines of evidence support the concept that the gut may be the reservoir for systemic sepsis and subsequent MOF in a number of pathophysiologic states.

Mol Biol Evol, 1998 Jul, 15(7), 871 - 9
Inferring pattern and process: maximum-likelihood implementation of a nonhomogeneous model of DNA sequence evolution for phylogenetic analysis; Galtier N et al.; A nonhomogeneous, nonstationary stochastic model of DNA sequence evolution allowing varying equilibrium G + C contents among lineages is devised in order to deal with sequences of unequal base compositions . A maximum-likelihood implementation of this model for phylogenetic analyses allows handling of a reasonable number of sequences . The relevance of the model and the accuracy of parameter estimates are theoretically and empirically assessed, using real or simulated data sets . Overall, a significant amount of information about past evolutionary modes can be extracted from DNA sequences, suggesting that process (rates of distinct kinds of nucleotide substitutions) and pattern (the evolutionary tree) can be simultaneously inferred . G + C contents at ancestral nodes are quite accurately estimated . The new method appears to be useful for phylogenetic reconstruction when base composition varies among compared sequences . It may also be suitable for molecular evolution studies.

Vet Immunol Immunopathol, 1998 May 15, 63(1-2), 123 - 9
Bystander stimulation of T cells in vivo by cytokines; Tough DF et al.; Immune responses to infectious agents, especially viruses, are often associated with extensive proliferation of T cells and transient enlargement of the lymphoid tissues . Since the precursor frequency of T cells for specific antigen is low, the bulk of the T cells proliferating in the primary response are presumably stimulated via non-antigen-specific mechanisms, e.g . via cytokines elicited by the infectious agent concerned . Such 'bystander' stimulation of T cells occurs in mice injected with agents that elicit production of type I interferon (IFN I) . Induction of IFN I in vivo causes marked stimulation of the CD44hi subset of CD8+ T cells and is prominent after injection of live viruses or products of bacteria such as lipopolysaccharide . Cytokines elicited by infectious agents may act as adjuvants during the primary response and could serve to boost the survival of long-lived memory cells.

Arch Inst Cardiol Mex, 1998 Jan-Feb, 68(1), 12 - 7
{Phenotype and reactivity of T-lymphocytes isolated from atheromatous plaque . Knowledge obtained from a transplant case}; Ramirez T et al.; The primary immunologic hypothesis assumes that the initial damage in atherosclerotic lesions is mediated by T lymphocytes reactive to heat shock proteins, lipoproteins, bacteria, virus or even donor MHC antigens . A frequent cause of heart transplant failure is the de novo formation of atheromatous lesions in the vessels of the transplanted organ despite their absence in the donor, thus suggesting that new lesions are secondary to a cellular immune response by the receptor . In this study we determined the phenotype and the reactivity of T cells from peripheral blood and from endomyocardial and atherectomy biopsies obtained from the same immunosuppressed patient who underwent a heart transplant in 1989 . A panel of homozygous HLA-typed, Epstein-Barr virus transformed B lymphocytes were used as stimulators in functional assays . Our results showed an important increase in the percentage of CD4+ cells in the atheromatous plaque as well as in the endomyocardium, and a considerable amount of TCR sigma+ lymphocytes in the atheromatous plaque . A considerable loss of alloreactivity to HLA antigens was also observed . These results suggest that although there are adequate conditions to mount a cellular immune response a state of cellular anergy exists towards HLA antigens probably as result of prolonged immunosuppressive therapy . The presence of obstructive lesions in this particular patient don't seem to be secondary to HLA alloreactivity but could be secondary to a switch in the cellular immune response as a consequence of chronic exposure to some donor antigen, thus explaining the increased proportion of TCR sigma+T lymphocytes.

Pneumologie, 1998 May, 52(5), 263 - 70
{Epidemiology, clinical aspects and prognosis of severe progressive community-acquired pneumonia}; Holtermann W et al.; BACKGROUND: Community-acquired pneumonia can lead to acute lung failure (parapneumonic ARDS) if the course is very severe . The clinical picture reflects a rapidly progressive and potentially fatal respiratory failure . Only occasional cases in which the clinical courses of community-acquired pneumonia lead to acute respiratory failure have been reported so far . The investigation was based on the observation that very severe progressive forms of community-acquired pneumonia are at present one of the most frequent conditions triggering ARDS . PATIENTS AND METHODS: A total of 66 patients of both sexes with an average age of 34 +/- 11 years were included in the retrospective investigation . The patients had been secondarily referred to the center for further treatment . After admission, the further course of the disease was recorded at five defined times (day of admission, 2nd day, 7th day, 14th day and day of spontaneous breathing or day of death) . The degree of disturbance of pulmonary function was registered with the scores of Morel and Murray . Further disorders of organ function were evaluated with the MOF score according to Goris, the "Definition Multiple disorder of Organ Function (DeMOF)" and the appraisal of the severity of the systemic inflammatory reaction with the sepsis score according to Elebute & Stoner . RESULTS: The duration of preclinical disease was 6 +/- 4 days and the duration of the pretreatment in the referring hospital was 10 +/- 10 days . A potential primary causative organism (bacteria n = 18, viruses n = 5, "atypical" pathogens n = 6, Candida species n = 4) could be isolated in 50% of the patients . A pre-existing underlying disease was found in 48% of cases . With a total lethality of 31%, this was affected neither by knowledge of the primary causative organism nor by previous diseases . The patients who died did so with improved lung function in a complete clinical picture of multiorgan failure . At the time of admission, 91% of the patients had severe ARDS (Morel III and IV) . An improvement of lung function could be demonstrated between the day of admission and the second day of treatment both with the score according to Morel and according to Murray (p < 0.05) . For the second day of treatment, a difference could be shown between the patients who survived and those who died (p < 0.05) . Owing to the systemic inflammatory reactions, a multiorgan functional disorder was found in 89% of the patients . There were the following findings with regard to the prognostic predictions from the score used: those who died and those who survived could be correctly differentiated with the DeMOF score from the 7th day of treatment and the sepsis from the 7th day of treatment and with the score of Goris from the 14th day of treatment after referral . CONCLUSIONS: The investigation proves that the most severe progressive forms of community-acquired pneumonia also occur both in patients who have previously appeared to be healthy and in younger patients . Despite the use of differentiated treatment measures, these illnesses are subject to a relatively high lethality . The results underscore the need for causal treatment of systemic inflammatory reaction, which is the most important problem in treatment of parapneumonic ARDS.

Z Gastroenterol, 1998 May, 36(5), 369 - 72
{Is chronic laryngitis associated with Helicobacter pylori? Results of a prospective study}; Jaspersen D et al.; H . pylori is found in the stomach of patients with chronic gastritis . The infection is usually transmitted by the gastro-oral route and bacteria could be identified in saliva and dental plaque . An essential cause of chronic laryngitis is gastroesophageal reflux . The aim of the study was to evaluate if a H.pylori-associated chronic laryngitis exists . 38 patients with chronic laryngitis underwent gastroscopy . Biopsies were taken from the gastric antrum and body, lower, middle and upper esophagus . H . pylori was diagnosed by rapid urease test and histology . 14 of the patients (36.8%) were H.pylori-positive, but the bacteria could not be identified between stomach and larynx . 24 patients were H . pylori-negative . Seven patients (18.4%) suffered from esophagitis, six of these patients were H . pylori-negative . The H . pylori-infected patients received triple therapy for one week, in case of esophogitis Omeprazole 20 mg BID was prescribed . Six weeks later a follow-up endoscopy was performed . The eradication rate was 12/14 (85.7%), in all patients with reflux the esophagitis was cured . The laryngitis was clinically and endoscopically unchanged in ten of the twelve (83.3%) patients after successful treatment for H . pylori; in the remaining two patients as well as in the two H . pylori-positive patients the laryngitis was improved . In six out of the seven patients with esophagitis the laryngitis had healed completely and was improved in the remaining patient . It may be concluded that there is no evidence for the existence of H . pylori-associated laryngitis, suggesting that acid reflux is the underlying etiology.

J Mol Biol, 1998 Jul 10, 280(2), 297 - 314
A common ancestor for oxygenic and anoxygenic photosynthetic systems: a comparison based on the structural model of photosystem I; Schubert WD et al.; The 4 A structural model of photosystem I (PSI) has elucidated essential features of this protein complex . Inter alia, it demonstrates that the core proteins of PSI, PsaA and PsaB each consist of an N-terminal antenna-binding domain, and a C-terminal reaction center (RC)-domain . A comparison of the RC-domain of PSI and the photosynthetic RC of purple bacteria (PbRC), reveals significantly analogous structures . This provides the structural support for the hypothesis that the two RC-types (I and II) share a common evolutionary origin . Apart from a similar set of constituent cofactors of the electron transfer system, the analogous features include a comparable cofactor arrangement and a corresponding secondary structure motif of the RC-cores . Despite these analogies, significant differences are evident, particularly as regards the distances between and the orientation of individual cofactors, and the length and orientation of alpha-helices . Inferred roles of conserved amino acids are discussed for PSI, photosystem II (PSII), photosystem C (PSC, green sulfur bacteria) and photosystem H (PSH, heliobacteria).Significant sequence homology between the N-terminal, antenna-binding domains of the core proteins of type-I RCs, PsaA, PsaB, PscA and PshA (of PSI, PSC and PSH respectively) with the antenna-binding subunits CP43 and CP47 of PSII indicate that PSII has a modular structure comparable to that of PSI .

FEBS Lett, 1998 May 29, 428(3), 123 - 6
Indirect evidence for structural changes coupled with QB- . formation in photosystem II; Reifarth F et al.; The thermal blockage of QA- . oxidation was analysed in PS II membrane fragments by monitoring flash-induced changes of the relative fluorescence quantum yield as a function of temperature . The results obtained reveal: (a) in dark-adapted samples the fraction of QA- . that is not reoxidised within a time domain of 10 s after the actinic flash increases with lowering the temperature (half-maximum effect at 250-260 K), (b) at low temperatures where QA- . generated in dark-adapted samples remains almost completely reduced, a significant extent of QA- . reoxidation arises when samples are used that were preilluminated at room temperature by one saturating flash followed by rapid freezing before performing the experiment, and (c) the extent of QA- . that is reoxidised at 258 K exhibits a characteristic binary oscillation as a function of the number of preillumination flashes given at room temperature . Based on these data it is inferred that QB and QB- . are located at different equilibrium positions in the QB site . As a consequence the formation of QB- . is coupled with significant structural changes that require sufficient flexibility of the protein matrix . This general feature corresponds with a recently proposed model for the acceptor side reactions of anoxygenic bacteria {Stowell, M.H.B., McPhillips, T.M., Rees, D.C., Soltis, S.M., Abresch, E . and Feher, G., Science 276 (1997) 812-816}.

Eur J Biochem, 1998 Apr 15, 253(2), 390 - 8
Different forms of the mRNA encoding the heat-shock transcription factor are expressed during the life cycle of the parasitic helminth Schistosoma mansoni; Lantner F et al.; Several cDNAs and a gene encoding the heat-shock transcription factor (HSF) of schistosome were cloned, and multiple forms of the mRNA were found at different developmental stages of the parasite . The encoded protein contained a DNA-binding domain with expected sequence identity (39-58%) to other HSF molecules, and two leucine zipper motifs (LZ123 and LZ4) involved in the oligomerization of HSF . Adult worms express three isoforms of HSF mRNA generated by alternative splicing inside the coding region that contains in-frame splice signals . Introns are not involved in the process since the deleted segments (36 bp or 45 bp) are not flanked by any intron in the gene . Structural variations generated by alternative splicing (insertion of 3 amino acids or 15 amino acids) are continual with LZ4 and added hydrophobic residues are in register with the hydrophobic heptad repeats of LZ4 . Structural diversity at the C-terminus of LZ4 may affect the strength of LZ4 interaction with the oligomerization domain (LZ123) and thus modulate the DNA-binding activity of HSF . The conservation of this mechanism in mouse and schistosome may reflect evolutionary pressure to generate multiple HSF species exhibiting functional diversity and capable of responding to different stress signals and physiological signals . Adult worms express HSF mRNA of 2.5 kb, in agreement with the size of the cDNA, while cercariae (developmental stage preceding adult worm) show multiple bands in the range 2.5-3 kb . Available data indicate that the HSF mRNAs of cercariae are inactive . We propose that these mRNA species are generated by an alternative splicing that incorporates introns, which inactivate the mRNA by the insertion of termination codons and/or by shifting of the reading frame . Parasite HSF protein produced in bacteria showed DNA sequence recognition similar to that of HSF in parasite extracts, i.e . the recombinant HSF reacted better with a variant heat-shock element (HSE; one base change in the third NGAAN pentamer of the ideal HSE consensus sequence) than with the ideal HSE . The size of the HSF gene is 12 kb and it is composed of ten exons and nine introns . Excluding the introns, the gene and cDNA show 100% sequence identity . A plant HSF gene contains only a single intron, which matches with the position of intron I2 of schistosome . That the position of this intron is conserved in remote species is indicative of an important function during evolution of the HSF gene.

Eur J Biochem, 1998 May 1, 253(3), 598 - 605
Engineering of factors determining alpha-amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1; Wind RD et al.; The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C) . Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft . All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved . Characterization of the F196G mutant CGTase of T . thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted {mutant F196G(delta'DE)} . This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa) . The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional C-terminal structure . The mutant showed a strongly reduced temperature optimum . Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced . Mutant F196G(delta'DE) displayed a strongly reduced raw-starch-binding capacity, similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain . Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase . Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides . These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

Dis Aquat Organ, 1998 May 14, 33(1), 25 - 31
Virulence and antigenic characteristics of a cultured Rickettsiales-like organism isolated from farmed Atlantic salmon Salmo salar in eastern Canada; Jones SR et al.; The present study describes culture, virulence and antigenic characteristics of a Rickettsiales-like organism (RLO) associated with mortality in farmed Atlantic salmon in eastern Canada . Clinical disease was reproduced in naive Atlantic salmon parr by intraperitoneal i.p . inoculation with kidney homogenate from naturally infected fish . Pure cultures of RLO were isolated into chinook salmon embryo (CHSE) cells from kidney of experimentally infected fish . The RLO caused cytopathic effect in cultured CHSE-214 typified by coalescing areas of swollen cells that eventually detached from the substrate . Bacteria in infected culture supernatants reacted with Piscirickettsia salmonis-specific polyclonal sera or monoclonal antibody (MAb) in an indirect fluorescent antibody test . IP inoculation with cultured RLO resulted in mortalities of 100, 62, 22.5 and 0% in Atlantic salmon, coho salmon, rainbow trout and common carp, respectively . Cultured RLO were sensitive to chloramphenicol, flumequine, oxytetracycline and oxolinic acid and insensitive to gentamicin and amphotericin B . RLO antigens were compared with those of 3 strains of P . salmonis from Chilean salmon by SDS-PAGE and immunoblotting . A silver-staining band of about 12 kDa was detected in proteinase K (PK) digests of all RLO strains, and a diffuse band of about 15 kDa was observed in 2 Chilean strains only . No other silver-stained bands were visible in PK digests of any strain examined . The polyclonal serum recognized 9 protein bands and multiple non-protein bands extending from less than 20 kDa to greater than 95 kDa in all isolates . The MAb reacted with an epitope in PK digests that occurred in all 4 strains on structures of widely ranging molecular masses, resulting in a ladder pattern similar to that obtained with polyclonal serum . Treatment of PK digests with periodic acid abolished reactivity with MAb and polyclonal serum . Co-elution of 2-keto-3-deoxyoctonate and MAb reactivity following size exclusion chromatography of solubilized P . salmonis suggested that the MAb recognized a lipopolysaccharide-associated epitope in all 4 RLO isolates . Cultural, virulence and antigenic similarities among the strains examined in the present study indicate that the eastern Canadian salmonid RLO should be considered a strain of P . salmonis.

Prof Nurse, 1998 May, 13(8), 504 - 7
Disinfection of latex gloves with ethyl alcohol; Grinnell F; The ability to disinfect latex gloves successfully between procedures would save time and be cost-effective . An in vitro study examined the efficacy of using an ethyl alcohol/bactericide compound to disinfect latex gloves contaminated with five common bacteria.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 7860 - 5
Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens; Lorito M et al.; Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus . The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato . High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development . Substantial differences in endochitinase activity were detected among transformants . Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A . solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani . The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria . These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.

J Biol Chem, 1998 Jul 10, 273(28), 17962 - 7
Structure-function relationships in OxlT, the oxalate/formate transporter of Oxalobacter formigenes . Topological features of transmembrane helix 11 as visualized by site-directed fluorescent labeling; Fu D et al.; Analysis of hydropathy suggests that in OxlT, the oxalate/formate antiporter of Oxalobacter formigenes, lysine 355 is within transmembrane helix no . 11 . To test this idea, we used single-cysteine, histidine-tagged OxlT variants to study the organization of a 30-residue segment (residues 344-373) containing this region . Topology was examined by probing the A345C and A370C proteins with Oregon Green maleimide carboxylic acid, an impermeant and fluorescent thiol-reactive agent . Examination of purified protein showed that only A370C was fluorescent after treating intact cells with the probe, while both proteins were modified in tests with isolated membrane ghosts . In addition, labeling of A370C, but not A345C, was blocked when external cysteines were protected with the impermeant and nonfluorescent agent, methanethiosulfonate ethyltrimethylammonium . These findings confirm that A345 faces the cytoplasm, while A370C faces the periplasm . A similar study focused on 13 single-cysteine variants positioned throughout the target segment . That work revealed a striking discontinuity in reactivity toward Oregon Green maleimide; cysteines within a 10-residue central core (residues 351-360) were not labeled when membranes were probed, but were readily modified after protein denaturation . We suggest this core resides within the lipid bilayer, unavailable to an impermeant reporter . Since this region includes position 355, we also suggest that lysine 355 lies within the OxlT hydrophobic sector, where it may facilitate the binding and translocation of the anionic substrates, oxalate and formate.

J Biol Chem, 1998 Jul 10, 273(28), 17539 - 43
Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase; Li QS et al.; We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings . The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts . More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties . These activities are characteristic of PNPase . Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity . We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme . The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W . (1996) EMBO J . 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study . We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts . The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 13398-13403).

J Clin Periodontol, 1998 May, 25(5), 394 - 8
Hyper-reactive peripheral neutrophils in adult periodontitis: generation of chemiluminescence and intracellular hydrogen peroxide after in vitro priming and FcgammaR-stimulation; Fredriksson M et al.; We have earlier reported a higher Fcgamma-receptor (FcgammaR)-mediated generation of reactive oxygen species, measured as luminol-enhanced chemiluminescence (CL) from peripheral neutrophils in adult periodontitis patients . The aims of this study were to confirm our previous results and to elucidate the mechanism of this phenomenon by measuring CL in parallel with the intracellular production of hydrogen peroxide, after stimulation with opsonized bacteria . To determine whether the higher CL was associated with altered responsiveness to priming, the cells were preincubated with tumor necrosis factor alpha (TNFalpha) and lipopolysaccharide (LPS) . While CL was significantly higher in subjects with periodontitis, there was no difference in hydrogen peroxide production between the patients and the controls, indicating that the hyperreactivity is related to the generation of other oxygen species than H2O2 and/or to processes in the outer cell membrane . The responsiveness to priming with LPS on CL was slightly but not significantly higher in the periodontitis group, suggesting that priming could be of value for distinguishing subjects with periodontitis . When assaying intracellular production of H2O2, TNFalpha and LPS had both a priming and an activating effect . There were no significant differences between the two groups . In conclusion, this study shows a higher FcgammaR-mediated CL of peripheral neutrophils from adult patients with periodontitis, thus confirming our earlier results . The hyperreactivity seems to be related to the outer cell membrane or to oxygen species other than H2O2.

Chemosphere, 1998 Jul, 37(2), 319 - 26
The 'two-phase closed bottle test'--a suitable method for the determination of 'ready biodegradability' of poorly soluble compounds; Richterich K et al.; The Two-Phase Closed Bottle test (BODIS test) is a cost-effective supplement of the existing OECD tests for ready biodegradability (OECD 301) due to its entire compatibility with them and its particular suitability for testing poorly soluble compounds . The comparison of a number of test data from this and other ready biodegradability tests showed that the BODIS test has a similar stringency in terms of the attainment of the pass level and the time window criterion as well . A significant influence of the strength of the bacterial inoculum on the test results was not observed.

Nucleic Acids Res, 1998 Jul 15, 26(14), 3358 - 63
Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity; Domachowske JB et al.; Eosinophil cationic protein (ECP) is one of two RNase A-superfamily ribonucleases found in secretory granules of human eosinophilic leukocytes . Although the physiologic function of eosinophils {and thus of the two eosinophil ribonucleases, ECP and eosinophil-derived neurotoxin (EDN)} remains controversial, we have recently shown that isolated human eosinophils promote ribonuclease-dependent toxicity toward extracellular virions of the single-stranded RNA virus, respiratory syncytial virus, group B (RSV-B) . We have also shown that recombinant human EDN (rhEDN) can act alone as a ribonuclease-dependent antiviral agent . In this work, we provide a biochemical characterization of recombinant human ECP (rhECP) prepared in baculovirus, and demonstrate that rhECP also promotes ribonuclease-dependent antiviral activity . The rhECP described here is N-glycosylated, as is native ECP, and has approximately 100-fold more ribonuclease activity than non-glycosylated rhECP prepared in bacteria . The enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor (RI) . Although rhECP was not as effective as rhEDN at reducing viral infectivity (500 nM rhECP reduced infectivity of RSV-B approximately 6 fold; 500 nM rhEDN, >50 fold), the antiviral activity appears to be unique to the eosinophil ribonucleases; no reduction in infectivity was promoted by bovine RNase A, by the amphibian ribonuclease, onconase, nor by the closely-related human ribonuclease, RNase k6 . Interestingly, combinations of rhEDN and rhECP did not result in either a synergistic or even an additive antiviral effect . Taken together, these results suggest that that the interaction between the eosinophil ribonucleases and the extracellular virions of RSV-B may be specific and saturable.

Scand J Gastroenterol, 1998 May, 33(5), 454 - 60
Gastric ulcer, atrophic gastritis, and intestinal metaplasia caused by Helicobacter pylori infection in Mongolian gerbils; Honda S et al.; BACKGROUND: Helicobacter pylori infection is associated with gastroduodenal disease in humans . In this study we aimed to show this relationship directly in Mongolian gerbils . METHODS: The animals were challenged orally with H . pylori and killed 1, 2, 3, and 6 months after inoculation for histologic and anti-H . pylori antibody titer examination . RESULTS: The spiral bacteria were observed in the mucus and gastric pits of all infected animals . A severe infiltration of the lamina propria by polymorphonuclear and mononuclear cells was seen 1 month after H . pylori inoculation . The submucosa was infiltrated by mainly mononuclear cells with formation of lymphoid follicles . Erosion of the gastric mucosa appeared soon after inoculation, whereas gastric ulcers, gastritis cystica profunda, and atrophy with goblet cell metaplasia occurred between 3 and 6 months after inoculation . In the duodenal mucosa a mild inflammatory cell infiltration with ballooning and diminished number of duodenal glands was seen . The IgG anti-H . pylori antibody titer increased gradually after 2 months of inoculation . CONCLUSIONS: Since the gastritis, gastric ulcers, atrophic gastritis, and intestinal metaplasia that developed in Mongolian gerbils were similar to those observed in humans, this model may be useful to study the therapy of gastric ulcer and, with a longer observation period, to confirm a possible relationship between H . pylori and malignancy.

Photochem Photobiol, 1998 Jun, 67(6), 700 - 13
Duck hepatitis B virus inactivation and 8-methoxypsoralen photoadduct formation in human platelet concentrates; Eble BE et al.; Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA) . To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus . A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5-6 log10 virus kill . The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium . Optimum conditions for PCI were 100 micrograms/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium . A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions . Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding . The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands . Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone . A minor subpopulation of DHBV was found to be somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.

Photochem Photobiol, 1998 Jun, 67(6), 683 - 99
The general kinetic model of electron transfer in photosynthetic reaction centers activated by multiple flashes; Shinkarev VP; A new general kinetic model for the functioning of photosynthetic reaction centers (RC) of purple bacteria, under multiple flash activation, has been developed . The model includes the primary electron donor (P870) as well as the primary (QA) and secondary (QB) acceptor quinones . The new features of this general model include: (1) consideration of four different states of the QB binding site (vacant, occupied by QB, by QB- and by QBH2), (2) incorporation of the dark relaxation of the RC between flashes, (3) the assumption of fast exchange of quinones between the RC and quinone pool in detergent micelles or chromatophore membrane, (4) description of the kinetics of electron transfer in both oxidized (no donor for P870+) and reduced (in the presence of donor for P870+) conditions simultaneously, (5) the consideration of both single and multiple flash activation of the RC of purple bacteria and (6) consideration of the cumulative effects of all previous flashes of the series in the response induced by the current flash . This model is used to calculate and predict (1) flash-induced binary oscillations of the secondary acceptor semiquinone (QB-), (2) flash-induced behavior of P870+ in the presence and absence of electron donor and (3) the apparent equilibrium constant of electron transfer between QA and QB and others . Different characteristics of RC are analyzed as a function of flash intensity, time between flashes, concentration of electron donor, redox-potential of the medium, concentration of pool quinone and quinol, association and dissociation equilibrium constants for quinone and quinol at the QB binding site, equilibrium constants of electron transfer between QA- and QB and between QA- and QB-, as well as the rate constants of oxidation of QA- and QB- by redox mediators . The proposed model can be used as a basis for assays of kinetic behavior of native and mutant RC of purple bacteria and for determination of the factors influencing the release of QH2 from RC . The latter is needed for analysis of factors controlling light-activated electron transport in the cytochrome bc1 complexes of purple bacteria by quinol molecules released from RC . The developed general approach for parallel consideration of flash-induced transitions of RC and its following dark relaxation between flashes can also be used for kinetic description of photosynthetic RC of oxygenic photosynthesis.

Bull Acad Natl Med, 1998, 182(2), 267 - 80; discussion 280-3
{Lyme borreliosis, emergent disease linked with the environment}; Perez-Eid C et al.; After a short historical presentation of the discovery of the pathogen and its vector, the authors present the current data on bacterial and acarologic taxonomy . Then they describe their results to assess the mechanisms of circulation of the bacteria in the forests of Ile-de-France, particularly in the forest of Rambouillet . The combined study of abundance and infection frequency of the vectors, small mammals and cervids leads to the characterization of periods and areas of higher risk . The risk periods correlate with high density of I . ricinus nymphs . The risk areas correspond to those of high density of cervids . The role of reservoir of small mammals is confirmed, to the one of large mammals, so debated, is clearly demonstrated.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2743 - 7
Importance of Extracellular Polymeric Substances from Thiobacillus ferrooxidans for Bioleaching; Gehrke T et al.; Leaching bacteria such as Thiobacillus ferrooxidans attach to pyrite or sulfur by means of extracellular polymeric substances (EPS) (lipopolysaccharides) . The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrochemical interaction with the negatively charged pyrite surface . EPS from sulfur cells possess increased hydrophobic properties and do not attach to pyrite, indicating adaptability to the substrate or substratum.

Appl Environ Microbiol, 1998 Jul 1, 64(7), 2572 - 7
Capillary Electrophoresis Measurements of Electrophoretic Mobility for Colloidal Particles of Biological Interest; Glynn JR Jr et al.; The electrophoretic mobilities of three bacterial strains were investigated by capillary electrophoresis (CE) and were compared with results obtained by microelectrophoresis (ME) . The CE measurements yielded bimodal electropherograms for two of the strains, thus illustrating for the first time that surface charge variations within a monoclonal population can be probed by CE . Intrapopulation variations were not detected by ME . The mobilities of three chemically distinct types of latex microspheres were also measured . Differences between the mean mobilities obtained by CE and ME were not statistically significant (P </= 0.50); the standard deviations of the CE measurements were typically 2 to 10 times smaller than those obtained by comparable ME measurements . The reproducibility of CE permitted batch-to-batch mobility variations to be probed for the bacteria (one of the strains exhibited such variations), and aggregation was evident in one of the latex suspensions . These effects were not measurable with ME.

Appl Environ Microbiol, 1998 Jul, 64(7), 2367 - 73
Isolation and entomotoxic properties of the Xenorhabdus nematophilus F1 lecithinase; Thaler JO et al.; Xenorhabdus spp . and Photorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the families Steinernematidae and Heterorhabditidae, respectively, were shown to produce different lipases when they were grown on suitable nutrient agar . Substrate specificity studies showed that Photorhabdus spp . exhibited a broad lipase activity, while most of the Xenorhabdus spp . secreted a specific lecithinase . Xenorhabdus spp . occur spontaneously in two variants, phase I and phase II . Only the phase I variants of Xenorhabdus nematophilus and Xenorhabdus bovienii strains produced lecithinase activity when the bacteria were grown on a solid lecithin medium (0.01% lecithin nutrient agar; 24 h of growth) . Five enzymatic isomers responsible for this activity were separated from the supernatant of a X . nematophilus F1 culture in two chromatographic steps, cation-exchange chromatography and C18 reverse-phase chromatography . The substrate specificity of the X . nematophilus F1 lecithinase suggested that a phospholipase C preferentially active on phosphatidylcholine could be isolated . The entomotoxic properties of each isomer were tested by injection into the hemocoels of insect larvae . None of the isomers exhibited toxicity with the insects tested, Locusta migratoria, Galleria mellonella, Spodoptera littoralis, and Manduca sexta . The possible role of lecithinase as either a virulence factor or a symbiotic factor is discussed.

Am J Gastroenterol, 1998 Jun, 93(6), 928 - 31
Ulcer recurrence after gastric surgery: is Helicobacter pylori the culprit?
Lee YT, Sung JJ, Choi CL, Chan FK, Ng EK, Ching JY, Leung WK, Chung SC.
OBJECTIVES: Helicobacter pylori is the most important cause of recurrent peptic ulcer disease . However, its role in ulcer recurrence after peptic ulcer surgery is unclear . We aimed at studying the prevalence and distribution of H . pylori in patients who had undergone peptic ulcer surgery, and any association between H . pylori infection and ulcer recurrence in these patients . METHODS: Patients with previous vagotomy or partial gastrectomy presenting with dyspepsia or ulcer bleeding were recruited . Ulcer recurrence was documented by endoscopy . Biopsy specimens were taken from the gastric remnant and gastroenteric anastomosis in patients with previous partial gastrectomy, or from the antrum and corpus in vagotomized patients . H . pylori infection was detected by either a positive rapid urease test or the presence of the bacteria on histology . RESULTS: Ninety-three patients were studied; 73 patients (78%) had partial gastrectomy and 20 (22%) had vagotomy with drainage . H . pylori infection was documented in 36 patients (49%) in the gastrectomy group and in 13 (65%) in the vagotomy group . Thirty-six patients in the gastrectomy group had recurrent ulcers and 15 (42%) of them had H . pylori infection . Twelve patients in the vagotomy group had recurrent ulcers and eight (67%) of them were H . pylori positive . The prevalence of H . pylori infection did not differ between patients with or without ulcer recurrence . CONCLUSION: H . pylori infection cannot account for ulcer recurrence after peptic ulcer surgery.

Annu Rev Biophys Biomol Struct, 1998, 27, 329 - 56
Cytochrome c oxidase: structure and spectroscopy; Michel H et al.; Cytochrome c oxidase, the terminal enzyme of the respiratory chains of mitochondria and aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water . Electron transfer is coupled to proton translocation across the membrane, resulting in a proton and charge gradient that is then employed by the F0F1-ATPase to synthesize ATP . Over the last years, substantial progress has been made in our understanding of the structure and function of this enzyme . Spectroscopic techniques such as EPR, absorbance and resonance Raman spectroscopy, in combination with site-directed mutagenesis work, have been successfully applied to elucidate the nature of the cofactors and their ligands, to identify key residues involved in proton transfer, and to gain insight into the catalytic cycle and the structures of its intermediates . Recently, the crystal structures of a bacterial and a mitochondrial cytochrome c oxidase have been determined . In this review, we provide an overview of the crystal structures, summarize recent spectroscopic work, and combine structural and spectroscopic data in discussing mechanistic aspects of the enzyme . For the latter, we focus on the structure of the oxygen intermediates, proton-transfer pathways, and the much-debated issue of how electron transfer in the enzyme might be coupled to proton translocation.

Sci Total Environ, 1998 Jun 18, 214, 1 - 10
Temporal changes of 210Po in temperate coastal waters; Wildgust MA et al.; The temporal variation of Polonium-210 (210Po) was examined in coastal sea water, the mussel Mytilus edulis, the winkle Littorina littorea and green alga Ulva lactuca in order to investigate the entry of 210Po into the marine food chain . More than 99% of 210Po in the water column occurred in the particulate phase . Dissolved 210Po concentrations peaked during the spring phytoplankton bloom and it is suggested this is related to preferential scavenging of 210Po by the increased numbers of bacteria, viruses and small dissolved particulates . Changes in L . littorea 210Po specific activity are thought not to be related to food, but to a drop in body weight following spawning . Much of the 210Po accumulated by M . edulis was located in the digestive gland . The specific activity of 210Po in the digestive gland of M . edulis was shown to be strongly correlated with changes in sea water suspended particulate specific activity . Examination of other trace metal (Ag, Al, As, Ca, Cd, Cr, Co, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Sb, Se, Sn and Zn) variations in the digestive gland revealed that class B and borderline metals had a strong positive correlation with 210Po . On-going work is investigating whether the accumulation and loss of 210Po is affected by the presence of metallothioneins.

J Chromatogr A, 1998 May 22, 807(2), 151 - 64
Trace chromatographic analysis of dimethyl sulfoxide and related methylated sulfur compounds in natural waters; Simo R; Dimethyl sulfoxide (DMSO) occurs in the environment as a result of a number of biogenic and anthropogenic production and emission processes . It is an environmentally significant compound because of its use as a substrate by bacteria and its potential role in the biogeochemical cycle of dimethyl sulfide (DMS), a climatically active trace gas . In this paper, current methods for DMSO determination at nanomolar levels in natural waters, all involving gas chromatography, are reviewed . Direct injection and separation of aqueous DMSO offers a simple and fast application, but exhibits limited sensitivity due to limitation on injection volumes . So far, most authors have preferred DMSO reduction and subsequent analysis of the evolved DMS by purge-and-trap preconcentration and flame photometric detection . Several reducing agents have been used, though some require cumbersome procedures or are very sensitive to operational conditions . The common algal component dimethylsulfoniopropionate (DMSP) acts as an interference in some reduction methods and, therefore, either DMSP elimination prior to DMSO analysis or correction a posteriori is required . DMSO can be analyzed along with DMS, methanethiol, dimethyl disulfide and DMSP in the same water sample, either sequentially or separately, so that comprehensive speciation of methylated sulfur is obtained . Owing to the biological activity of DMSO, appropriate water sampling and handling procedures must be applied . Acidification and freezing appear to be suitable for aqueous DMSO storage, although immediate analysis in the field is always preferable . Future directions of DMSO determination in aquatic environments are suggested.

Environ Health Perspect, 1998 Jun, 106 Suppl 3, 841 - 7
Prenatal methylmercury exposure and children: neurologic, developmental, and behavioral research; Myers GJ et al.; Mercury is present in the earth's crust and is methylated by bacteria in aquatic environments to methylmercury (MeHg) . It is then concentrated by the food chain so predatory fish and sea mammals have the highest levels . Thus, consuming seafood leads to exposure . MeHg readily crosses the placenta and the blood-brain barrier and is neurotoxic . The developing fetal nervous system is especially sensitive to its effects . Prenatal poisoning with high dose MeHg causes mental retardation and cerebral palsy . Lower level exposures from maternal consumption of a fish diet have not been consistently associated with adverse neurodevelopmental outcomes . However, most studies have considerable uncertainty associated with their results . Two large controlled longitudinal studies of populations consuming seafood are underway that are likely to determine if any adverse effects can be identified . No adverse associations have been found in the Seychelles, where exposure is mainly from fish consumption . In the Faroe Islands where exposure is primarily from consumption of whale meat and not fish, adverse associations have been reported . The Seychelles population consumes large amounts of marine fish containing MeHg concentrations similar to commercial fish in the United States . Current evidence does not support the hypothesis that consumption of such fish during pregnancy places the fetus at increased neurodevelopmental risk.

Transplantation, 1998 Jun 15, 65(11), 1515 - 9
Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents; Kearns-Jonker M et al.; BACKGROUND: The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents . Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations . METHODS: We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens . Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses . RESULTS AND CONCLUSIONS: VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.

Trends Biochem Sci, 1998 Jun, 23(6), 208 - 12
Ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome; Ramakrishnan V et al.; Models of the bacterial ribosome based on recent structural analyses are beginning to provide new insights into the protein synthetic machinery . Central to evolving models are the high-resolution structures of individual ribosomal proteins, which represent detailed probes of their local RNA and protein environments . Ribosomal proteins are extremely ancient molecules; the structures therefore also provide a unique window into early protein evolution . Many of the proteins contain domains that are present in more recently evolved families of RNA- and DNA-binding proteins . Such structural homology can be used to predict mechanisms by which proteins interact with RNA in the ribosome.

Lin Chuang Er Bi Yan Hou Ke Za Zhi, 1997 Mar, 11(3), 112 - 4
{Infective factors of adult secretory otitis media}; Lin G et al.; There were histories of the upper expiratory tract infection in 49 out of 86 cases in adult secretory otitis media (SOM) . Among them, thirty two cases were administrated antibiotics a week ago when the effusions were collected . The patient's Eustachian tubes in twenty nine cases were clinically ventilated . The endotoxins were positive in 39 out of 86 specimen tested with limulus assay, among these specimen, bacteria were cultured from 11 specimen . The data exibited that the appearance of the SOM is related to the existence of the infective factors in the middle ear cavity . The upper expiratory tract infection before the onset of the middle ear effusions is one of the important causes inducing the disease . The lower rate of bacteria culture than that of endotoxins is related with the administration of antibiotics before the onset of the disease . The administration of antibiotics in the treatment of the middle ear effusions will help to elimilate bacteria in the cavity of the middle ear effusions, improve the ventilating function of the Eustachian tube and make the effusions turn to disappearance.

J Nat Prod, 1998 Jun 26, 61(6), 857 - 8
Isolation of 2-(3'-bromo-4'-hydroxyphenol)ethanamine from the New Zealand ascidian Cnemidocarpa bicornuta; Lindsay BS et al.; From the ascidian Cnemidocarpa bicornuta, 2-(3'-bromo-4'-hydroxyphenol)ethanamine (3'-bromotyramine) (1) has been isolated along with the previously reported sponge metabolite, 1,3-dimethylisoguanine . The structure of 1 was confirmed by synthesis.

Pathology, 1998 May, 30(2), 173 - 6
Effect on organism recovery rate from BacT/Alert blood cultures with reduced incubation period; Mukerjee C et al.; This retrospective study evaluated 15,377 sets of BacT/Alert blood cultures to determine incubation time for blood cultures . Ninety-six per cent (1476) of total isolates signalled positive within five days and 56 isolates turned positive in five to seven days . Of the 56 organisms recovered between five and seven days, 49 were considered contaminants and seven were considered clinically significant . On assessing the medical records of the patients with the seven clinically significant isolates, it was determined that the clinical outcome would not have changed if these isolates were missed . We conclude that a five day incubation protocol reduces the recovery of skin contaminants while not significantly decreasing the recovery of clinically significant organisms . The data suggest that the incubation time can be further reduced but this policy will depend on the individual institution and their patient population mix.

Kansenshogaku Zasshi, 1998 May, 72(5), 487 - 92
{The role of heat shock protein 60 (HSP60) of Helicobacter pylori in adhesion of H . pylori to human gastric epithelial cell}; Yamaguchi H et al.; Adhesion of Helicobacter pylori to both human gastric carcinoma cell lines (MKN45, MKN28 and KATO III) and prepared primary human gastric epithelial cells were analyzed with flow-cytometry . All strains adhered to human gastric carcinoma cells . Especially, these strains strongly adhered to MKN45 cells . Adhesion of H . pylori strains to prepared primary human gastric epithelial cells was also observed . However, the adherence rates of H . pylori to these cells were different among the cells used . These results suggested that the host factor might be important for adhesion of the bacteria to human gasgric cells . In addition, H20 monoclonal antibody directed to H . pylori HSP60 inhibited the adhesion of H . pylori to both cells . These results indicate that H . pylori HSP60 might be associated with the adhesion of the bacteria to human gastric epithelial cells.

J Mol Biol, 1998 Jun 12, 279(3), 605 - 19
Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans; Smith HE et al.; In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod . Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP) . In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments . Filaments composed of MSP dimers are thought to provide the motive force . We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation . Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct . A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP . Single missense mutations were identified and mapped to the crystal structure of A . suum MSP . Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface . Multiple additional mutations were obtained in the two carboxy-terminal beta strands, a region not predicted to be involved in protein folding or dimer formation . Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation . A number of compensating mutations that restore the interaction also map to this region . The data suggest that the carboxy-terminal beta strands are directly involved in interactions required for MSP filament assembly.

J Endod, 1998 Feb, 24(2), 112 - 5
In vitro adhesion of two strains of Prevotella nigrescens to the dentin of the root canal: the part played by different irrigation solutions; Calas P et al.; Blocks of bovine incisor dentin, on the root canal surface of which a smear layer had been formed, were inoculated in vitro with two strains of Prevotella nigrescens, a wild sampled strain and a reference one (NCTC 9336) . Half the blocks were pretreated with irrigating solutions: 6% citric acid for 5 min + 6.25% sodium hypochlorite for 10 min . They were compared with the other blocks simply rinsed in distilled water (i.e . the control samples) . The bacteria adhering to the dentin surface after an incubation time of 3 h were counted by direct examination using a scanning electron microscope . The adhesion of P . nigrescens was less marked on all of the samples treated with irrigating solutions . Adherence was particularly significant in the case of the wild strain (F = 10.22) . The latter was far more active than the reference strain (F = 35.82) . The use of a chelating agent at the end of root canal preparation served to remove the smear layer and limited the attachment of P . nigrescens to the dentin.

Curr Opin Cell Biol, 1998 Jun, 10(3), 317 - 22
SMC protein complexes and higher-order chromosome dynamics; Hirano T; The structural maintenance of chromosome (SMC) family of proteins represents an expanding group of chromosomal ATPases that are highly conserved among Bacteria, Archaea and Eukarya . During the past year, significant progress has been made towards understanding the cellular functions and molecular activities of this new class of proteins . Emerging evidence suggests that eukaryotic SMC proteins form large protein complexes with non-SMC subunits and act as key components for a wide variety of higher-order chromosome dynamics.

Curr Opin Cell Biol, 1998 Jun, 10(3), 311 - 6
DNA mismatch repair and cancer; Prolla TA; Mutations in DNA mismatch repair (MMR) genes have been associated with hereditary nonpolyposis colorectal cancer . Studies in bacteria, yeast and mammals suggest that the basic components of the MMR system are evolutionarily conserved, but studies in eukaryotes also imply novel functions for MMR proteins . Recent results suggest that mutations in MMR genes lead to tumorigenesis in mice, but DNA replication errors appear to be insufficient to initiate intestinal tumorigenesis in this model system . Additionally, MMR-deficient cell lines display a mutator phenotype and resistance to several cytotoxic agents, including compounds widely used in cancer chemotherapy.

Immunology, 1998 Mar, 93(3), 323 - 8
In vivo exposure to Porphyromonas gingivalis up-regulates nitric oxide but suppresses tumour necrosis factor-alpha production by cultured macrophages; Frolov I et al.; The present study was designed to test whether the functional response of mouse macrophages elicited by chronic exposure to bacteria will be different from that of cells elicited by a non-bacterial irritant . Macrophage elicitation was conducted by Porphyromonas gingivalis, a major periodontal pathogen, in comparison to a standard elicitation by thioglycollate (TG) . We measured lipopolysaccharide (LPS)-induced nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) secretion by the elicited macrophages, and the expression of inflammatory cytokines in the whole elicited cell population . In addition, we tested the response of TG-elicited macrophages to pretreatment with P . gingivalis LPS in vitro . Mouse peritoneal macrophages were harvested 4 days after intraperitoneal injection of TG or heat-killed P . gingivalis . TG-elicited macrophages produced undetectable levels of TNF-alpha and approximately 0.5 microM of NO . The stimulation of the macrophages with LPS resulted in the secretion of NO and TNF-alpha in a dose-dependent manner . The P . gingivalis-elicited macrophages produced basal levels of approximately 5 microM NO, but TNF-alpha was not detectable . LPS stimulation of these cells further increased the secretion of NO eightfold while TNF-alpha remained undetectable . The NO secretion by P . gingivalis-elicited cells was significantly higher than that by TG-elicited cells . Examination of cytokine expression in the whole elicited cell population revealed that both P . gingivalis-elicited cells and TG-elicited cells expressed messenger RNA for interleukin-2 (IL-2), TNF-alpha and interferon-gamma (IFN-gamma), but not for IL-4 . IL-6 was expressed in P . gingivalis-elicited cells only . Pretreatment of TG-elicited macrophages with P . gingivalis LPS for 24 hr prior to a second LPS challenge resulted in down-regulation of TNF-alpha secretion and up-regulation of NO secretion, a response similar to that seen in P . gingivalis-elicited peritoneal macrophages . The results suggest that the in vivo exposure of resident macrophages to P . gingivalis induces functional changes in peritoneal macrophages . These changes might be due to the effect of P . gingivalis LPS.

Microbiology, 1998 Jun, 144 ( Pt 6), 1537 - 47
Characterization of the glnB gene product of Nostoc punctiforme strain ATCC 29133: glnB or the PII protein may be essential; Hanson TE et al.; Bacterial PII proteins, encoded by glnB genes, are central signalling molecules in nitrogen regulatory pathways and are modulated by post-translational modification in response to the cellular nitrogen status . The glnB gene was cloned from the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme strain ATCC 29133 (PCC 73102) by heterologous hybridization to a Synechococcus sp . strain PCC 7942 gene fragment . Expression of the cloned gene was verified by hybridization to N . punctiforme total RNA and a single cross-reactive polypeptide was observed in immunoblots of N . punctiforme extracts probed with anti-Synechococcus 7942 PII antiserum . Modification of the purified N . punctiforme PII protein by a Synechococcus 7942 PII kinase was observed, but modified forms of PII were not detected in extracts of N . punctiforme from a variety of incubation conditions . The N . punctiforme glnB gene could not be disrupted by targeted gene replacement unless a second copy of glnB was provided in trans, suggesting that the gene or gene product is essential for growth under the conditions tested.

Microbiology, 1998 Jun, 144 ( Pt 6), 1527 - 35
Lipopolysaccharide expression within the genus Bordetella: influence of temperature and phase variation; van den Akker WM; LPSs play an important role in bacterial pathogenesis . In this study, the LPS expression of the seven known Bordetella species and its dependency on growth temperature was analysed by oxidative silver staining of proteinase-K-treated whole bacteria separated by Tricine-SDS-PAGE . The bordetellae were found to have extensively variable LPS in a species-specific way . In addition, the human and ovine Bordetella parapertussis strains exhibited host-specific LPS expression . LPSs from human B . parapertussis strains grown at 37 and 25 degrees C were distinct . Growth temperature also affected LPS production by several Bordetella bronchiseptica strains . In some of these cases, BvgAS, the global regulator of virulence factors, was involved in this regulation of LPS biosynthesis . In contrast, no evidence was found for the involvement of the Bordetella pertussis BvgAS system in regulation of LPS synthesis . The obligate human pathogens B . pertussis and Bordetella holmesii are closely related but were shown to produce immunologically distinct LPSs . These species are isolated from the upper respiratory tract and blood, respectively . This raises several interesting questions concerning the potential role of LPS as a virulence factor in the infection processes.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1998 Jun, 85(6), 720 - 5
Development of periradicular lesions in immunosuppressed rats; Waterman PA Jr et al.; PROBLEM: The role of bacteria has been well established in pulpal and periapical diseases, but the contribution of the host defenses is less clear . OBJECTIVES: The purpose of this study was to compare periradicular lesion development in immunosuppressed rats with that in normal rats . STUDY DESIGN: Fifteen rats were given weekly injections of Cytoxan (Bristol Laboratories) to suppress their immune systems . The pulps of mandibular first molars of these animals and another 15 rats that had received no medications were exposed and left open to their oral flora . The rats were killed at 2, 4, and 6 weeks . Radiographic analysis was performed by means of a computer linked to a digitizing board and stylus . In addition, specimens were decalcified, sectioned, stained, and examined under a microscope with a grid to quantify relative percentages of surface areas of bone, root, periodontal ligament, marrow spaces, soft tissue, and inflammatory infiltrate . RESULTS: Statistical analysis showed a significantly greater radiographic bone loss in the immunosuppressed group only at 4 weeks . No significant histologic differences were found between the two groups . CONCLUSION: Our results suggest that reduction of circulating leukocytes may not significantly affect the development of periradicular pathosis in rats.

Glycobiology, 1998 Aug, 8(8), 791 - 8
Coordinated binding of sugar, calcium, and antibody to macrophage C-type lectin; Hosoi T et al.; Mouse macrophage galactose/N-acetylgalactosamine-specific C-type lectin (MMGL) is a type II transmembrane glycoprotein belonging to the C-type lectin family . Our development of monoclonal antibodies led us to discover that a calcium-dependent conformational change is detected by an antibody (termed mAb LOM-11) and that the antibody's binding to the respective site locks the lectin in an active conformation . These findings correspond to the divalent cation-mediated regulatory mechanisms in a family of cell adhesion molecule integrins that have gained much attention . We now provide direct evidence that mAb LOM-11 increases the affinity of the lectin for calcium ions as a mechanism for the conformational lock using a soluble recombinant form of MMGL (rML) produced in bacteria . Furthermore, we discovered by using an enzyme-linked immunosorbent assay that specific monosaccharides induced a binding site for mAb LOM-11 on the immobilized rML under low calcium environments . We also demonstrated that cell surface MMGL on a transfectant cell line underwent a conformational change upon addition of calcium or ligands, as detected by the binding of mAb LOM-11 . These properties are reminiscent of ligand-induced binding sites defined for integrins . The present results suggest a possibility that the mAb LOM-11 binding site on the lectin may be a site at which protein-protein interaction helps to fine tune the specificity of the C-type lectins by means of coordinated recognition mechanisms.

Rev Sci Tech, 1998 Apr, 17(1), 351 - 64
Lessons from gene knockouts; Osterrieder N et al.; The authors describe the technique for the application of homologous recombination in embryonic stem cells, which is now widely used to engineer mice which carry specific knockouts of genes . A summary is given of some of the knowledge of the pathogenesis of and resistance to infections with parasites, bacteria, or viruses which has accumulated during recent years, based on the investigation of knockout mice . Special emphasis is placed on knockout animals which lack components of the cytokine network, lack genes which are critical for the correct presentation of antigens or are deficient in different immune cell subsets . In addition, a brief explanation is offered of the possibilities for inducing targeted deletions or mutations in genes of livestock species (e.g., by nuclear transfer or by mutagenesis using the alkylating agent N-ethyl-N-nitrosourea) which could lead to the breeding of animals which are resistant to infectious diseases in the future.

Am J Respir Crit Care Med, 1998 Jun, 157(6 Pt 1), 1967 - 74
Entry and intracellular growth of Legionella dumoffii in alveolar epithelial cells; Maruta K et al.; We have found that Legionella dumoffii strain Tex-KL (ATCC 33343) invades into and proliferates in the human lung alveolar epithelial-cell line A549 in vitro . The organism associated with the A549 cells at a 10-fold greater magnitude than L . pneumophila Philadelphia-1 during in vitro coculture for 1 h . Thereafter, L . dumoffii Tex-KL invaded the cells at a significantly higher rate (100- to 1,000-fold) than did L . pneumophila Philadelphia-1 . After internalization, however, both bacteria proliferated at the same rate . This in vitro finding led us to examine the bacterial localization in lungs in a fatal case of L . dumoffii pneumonia . Double immunostaining revealed the bacteria in surfactant apoprotein A-positive cells (i.e., type II alveolar epithelial cells) . Next, we infected guinea pigs intratracheally with L . dumoffii Tex-KL . The animals became sick with a fever from 24 h to 48 h after infection with 10(4) to 10(9) cfu of L . dumoffii Tex-KL . The lung tissues were examined through electron microscopy at definite intervals . Many bacteria were found not only inside phagocytic cells in the alveolar space, but also in type I and type II alveolar epithelial cells . These findings strongly suggest that L . dumoffii has an ability to invade into and proliferate in human alveolar epithelial cells, which may explain the rapid and fulminant progress of pneumonia caused by L . dumoffii.

Am J Respir Crit Care Med, 1998 Jun, 157(6 Pt 1), 1951 - 8
Bronchial artery embolization for the treatment of hemoptysis in patients with cystic fibrosis; Brinson GM et al.; Hemoptysis is common in patients with cystic fibrosis (CF) . Bleeding may vary in severity, ranging from minor blood-streaking of sputum to expectoration of significant quantities of blood . Major hemoptysis, defined as bleeding greater than 240 ml/24 h, represents a medical emergency . Bronchial artery embolization (BAE) is one of the treatment options for hemoptysis . We reviewed the 10-yr experience at the University of North Carolina Hospitals in the treatment of hemoptysis by BAE . Eighteen patients with CF were hospitalized on 29 occasions and underwent 36 BAE procedures for the control of hemoptysis . Most patients (n = 11) had very severe lung disease (FEV1 < 35%) with a high incidence (n = 9, 50%) of multi-drug-resistant bacteria . Fifteen patients (n = 33 procedures) were followed for a mean of approximately 22 mo after BAE . The overall efficacy of BAE for initial control of hemoptysis was 75% (n = 22) after one session, 89% (n = 26) after two sessions, and 93% (n = 27) after three sessions . The overall recurrence rate per episode was 46% (12/26 presentations in four patients) with a mean time for recurrence of approximately 12 mo . There was a high incidence (75%) of bleeding from nonbronchial systemic collateral vessels among patients (n = 7) who had undergone a previous BAE . There were two deaths associated with massive hemoptysis despite BAE . Three patients had transient neurologic deficits during BAE . We concluded that BAE is a relatively safe and effective means of treating significant hemoptysis in patients with CF.

Biochem Biophys Res Commun, 1998 Jun 9, 247(1), 181 - 5
Chiral inversion of 1-hydroxyethylpyrene enantiomers mediated by enantioselective sulfotransferases; Landsiedel R et al.; The benzylic alcohol 1-hydroxyethylpyrene (1-HEP) is activated to a mutagen by sulfotransferases . The sulfuric acid ester formed is difficult to detect, as it is rapidly hydrolysed back to the alcohol . Incubation of the individual enantiomers of 1-HEP with human hydroxysteroid sulfotransferase (hHST) or estrogen sulfotransferase (hEST), expressed in bacteria, led to the formation of the other enantiomer . The rates of sulfation were determined from the initial rates of chiral inversion of the alcohol, knowing that hydrolysis follows an SN1 mechanism and therefore produces racemic alcohol . hEST showed high enantioselectivity for S-1-HEP, whereas hHST strongly preferred the R-enantiomer . The rates of sulfation of the preferred enantiomers were high, similar to those for the prototype substrates of hEST (beta-estradiol) and hHST (dehydroepiandrosterone) . Moreover, after a 30-min incubation of S-1-HEP with hEST, 95% of the recovered alcohol showed the R-configuration, indicating that several cycles of sulfation and hydrolysis had led to the depletion of one enantiomer and to the enrichment of the other enantiomer.

Biochem Biophys Res Commun, 1998 Jun 9, 247(1), 129 - 35
Novel carbazole degradation genes of Sphingomonas CB3: sequence analysis, transcription, and molecular ecology; Shepherd JM et al.; The degradation of aromatic compounds by bacteria is dependent upon specific catabolic operons . The unique car locus isolated from Sphingomonas CB3 encodes the first four enzymes involved in the catabolism of the azaarene carbazole . These include a class II three-component dioxygenase enzyme system, a dihydrodiol dehydrogenase, an extradiol (meta-cleavage) dioxygenase, and a hydrolase . Homology of deduced amino acid sequences is closer to corresponding biphenyl catabolic genes than to previously characterised carbazole degradation genes . Gene arrangement is also identical to that found in some bph loci . The car genes are transcribed when carbazole is utilised as a sole carbon source, and although biphenyl does not serve as a growth substrate for Sphingomonas CB3 it is able to act as a non-metabolisable inducer of the car locus . Ecologically the car genes were detected in polycyclic aromatic hydrocarbon (PAH) contaminated soil associated with a former town gas site.

Biotechnology (N Y), 1995 Dec, 13(13), 1474 - 8
The yeast tribrid system--genetic detection of trans-phosphorylated ITAM-SH2-interactions; Osborne MA et al.; Protein-protein interactions are often dependent on the post-translational modification of one component of a complex . To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system . We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate . Using the gamma subunit of the high-affinity IgE receptor, Fc epsilon RI, this approach has been used to isolate a novel SH2-containing family member . The mRNA encoding this novel protein is differentially expressed in rat tissues . The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7825 - 9
Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis; Gamble RL et al.; ETR1 represents a prototypical ethylene receptor . Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants . The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization . The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria . The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified . Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP . The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine . Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain . Truncations were used to delineate the region required for histidine kinase activity . An examination of cation requirements indicated that ETR1 requires Mn2+ for autophosphorylation . These results demonstrate that higher plants contain proteins with histidine kinase activity . Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.

Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7396 - 401
Trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein; Cordfunke R et al.; The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a "locked" chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II) . These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket . Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle . Surprisingly, both are able . We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy . Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics . We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

Cell Tissue Res, 1998 Jun, 292(3), 597 - 607
Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller); Flowers AE et al.; The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular . Archaeocytes and choanocytes are the major sponge cell types present . Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities . The protocol also has the advantage of separating broken from intact cells of O . spongeliae . The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells . Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association . However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines . The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Nat Biotechnol, 1996 Nov, 14(11), 1557 - 61
The extreme sensitivity of Mycobacterium tuberculosis to the front-line antituberculosis drug isoniazid; Deretic V et al.; Mycobacterium tuberculosis is a natural mutant in oxyR, a close homolog of the central regulator of peroxide stress response in enteric bacteria . Inactivation of oxyR is specific for M . tuberculosis and other members of the M . tuberculosis complex . This phenomenon appears as a paradox due to the ability of this organism to parasitize host macrophages, in which the ingested organisms are likely to be exposed to reactive oxygen intermediates . However, the surprising finding that M . tuberculosis has multiple deletions, nonsense and frameshift mutations in oxyR may help explain the exceptionally high sensitivity of M . tuberculosis to the potent antituberculosis agent isoniazid . One of the genes affected by oxyR lesions, ahpC (encoding an alkylhydroperoxide reductase) may determine the intrinsic sensitivity of mycobacteria to isoniazid.

J Biol Chem, 1998 Jun 19, 273(25), 15404 - 11
The two {4Fe-4S} clusters in Chromatium vinosum ferredoxin have largely different reduction potentials . Structural origin and functional consequences; Kyritsis P et al.; The 2{4Fe-4S} ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria . The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV . This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters . The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials . A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of {4Fe-4S} clusters in proteins . (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C . vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster . (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines . These findings set the framework in which previous observations on ferredoxins can be better understood . They also shed light onto the possible occurrence and properties of very low potential {4Fe-4S} clusters in less well characterized proteins.

J Appl Microbiol, 1998 Apr, 84(4), 523 - 30
The microstructure and distribution of micro-organisms within mature Serra cheese; Parker ML et al.; The distribution of micro-organisms in mature Serra, a traditional Portuguese cheese made from unpasteurised ewes' milk without added starter culture, was examined by light microscopy and electron microscopy . Four populations of micro-organisms were recognized according to their position within the cheese: (i) those present as apparently axenic colonies within the curd matrix; (ii) bacteria growing along curd junctions; (iii) yeasts and bacteria present in the smear on the surface of the cheese and (iv) bacteria found in cracks which penetrated the outer part of the cheese from the rind . Two types of crystals were observed, together with contaminants of vegetable origin and somatic cells originating from the milk.

Transfusion, 1998 May, 38(5), 424 - 8
Effect of 24-hour storage at 25 degrees C on the in vitro storage characteristics of CPDA-1 packed red cells; Ruddell JP et al.; BACKGROUND: Packed red cells (RBCs) warmed above 10 degrees C are generally discarded . Few data exist on the degree of accelerated metabolism and increased hemolysis of packed RBCs allowed to warm . STUDY DESIGN AND METHODS: Twenty-four CPDA-1 packed RBC units were combined in 3-unit pools and subdivided into 2 test units and a control unit . One test unit from each pool was warmed to 25 degrees C for 24 hours on Day 6 and the other test unit was warmed on Day 20; control units were maintained at 1 to 6 degrees C . RBC and supernatant chemistries and RBC morphology were measured weekly (Days 0, 7, 14, 21, and 28) and on the day before warming (Days 6 and 20) . RESULTS: Warming CPDA-1 packed RBCs accelerated the catabolism of glucose 10-fold and produced concentrations of glucose, lactate, and ATP after 25 days of storage that were equivalent to those in unwarmed units at 35 days . Supernatant sodium and potassium concentrations were corrected partially with warming . RBC morphology transiently normalized with warming and without increased hemolysis; no bacteria growth was detected . CONCLUSION: One day of 25 degrees C storage of CPDA-1 packed RBCs accelerates essential metabolite break-down equivalent to 10 days of storage at 1 to 6 degrees C . It does not appear to matter whether the packed RBCs are warmed on Day 6 or Day 20 . This information may be useful in determining the acceptability of blood allowed to warm above 10 degrees C.

J Biol Chem, 1998 Jun 26, 273(26), 15906 - 12
Grb10 identified as a potential regulator of growth hormone (GH) signaling by cloning of GH receptor target proteins; Moutoussamy S et al.; The cloning of receptor targets procedure, used so far to identify proteins associated with tyrosine kinase receptors was modified to clone SH2 proteins able to bind to the growth hormone receptor (GHR) . The cytoplasmic region of GHR, a member of the cytokine receptor superfamily does not contain tyrosine kinase activity . It was thus phosphorylated in bacteria by the Elk tyrosine kinase and radiolabeled to screen a mouse expression library . With this probe, we identified Shc and the p85 subunit of phosphatidylinositol 3-kinase as direct targets of the receptor . The other proteins identified, Csk, Shb, Grb4, and Grb10 are new potential transducers for cytokine receptors . We show in Huh-7 hepatoma cells that Grb10 and GHR associate under GH stimulation . Co-transfections in 293 cells further show that Grb10 interacts with both the GHR and Jak2 . Functional tests demonstrate that Grb10 inhibits transcription of two reporter genes containing, respectively, the serum response element of c-fos and the GH response element 2 of the Spi2.1 gene, whereas it has no effect on a reporter gene containing only Stat5 binding elements . Our results suggest that Grb10 is a new target for a member of the cytokine receptor family that down-regulates some GH signaling pathways downstream of Jak2 and independently of Stat5.

Biochemistry, 1998 May 26, 37(21), 7725 - 32
A source of response regulator autophosphatase activity: the critical role of a residue adjacent to the Spo0F autophosphorylation active site; Zapf J et al.; Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes . The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes . Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation . The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined . Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54 . For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min) . It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis . It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA . Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position . It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.

Biochemistry, 1998 May 26, 37(21), 7696 - 707
Relationship between enzyme specificity and the backbone dynamics of free and inhibited alpha-lytic protease; Davis JH et al.; To better understand the structural basis for the observed patterns in substrate specificity, the backbone dynamics of alpha-lytic protease have been investigated using 15N relaxation measurements . The enzyme was inhibited with the peptide boronic acid N-tert-butyloxycarbonyl-Ala-Pro-boroVal {Kettner, C . A., et al . (1988) Biochemistry 27, 7682}, which mimics interactions occurring in the tetrahedral transition state or nearby intermediates, and the dynamics of the unbound and inhibited enzyme were compared . Arrayed 2-D NMR spectra were acquired to measure T1, T2, and steady-state inverted question mark1H inverted question mark-15N NOE of >95% of the backbone amides in both protein samples . The overall rotational correlation time tauc was found to be 8.1 ns . Values of the spectral density function J(omega) at omega = 0, omegaN, and approximately omegaH were derived from the relaxation results using reduced spectral density mapping {Ishima, R., & Nagayama, K . (1995) Biochemistry 34, 3162} . The resultant spectral densities were interpreted to indicate regions of fast motion (nanosecond to picosecond) and of intermediate chemical exchange (millisecond to microsecond) . The protein has 13 regions with increased motion on the fast time scale; these generally fall on exterior turns and loops and most correlate with regions of higher crystallographic B-factors . Several stretches of backbone undergo intermediate chemical exchange, indicating motion or other processes that cause temporal chemical shift changes . A comparison of spectral densities for both the free and inhibited enzymes revealed that inhibitor binding preferentially stabilizes regions undergoing chemical exchange (which predominate around the active site) and only minimally affect regions of rapid motion . Slow motions, suggestive of backbone plasticity, are observed in most of the binding pocket residues . This may point to a mechanism for the observed broad specificity of the enzyme . The significance of the observed dynamics for substrate binding and specificity is discussed.

J Mol Biol, 1998 May 8, 278(3), 655 - 66
Protein folding and protein evolution: common folding nucleus in different subfamilies of c-type cytochromes?
Ptitsyn OB.
Amino acid sequences of seven subfamilies of cytochromes c (mitochondrial cytochromes c, c1; chloroplast cytochromes c6, cf; bacterial cytochromes c2, c550, c551; in total 164 sequences) have been compared . Despite extensive homology within eukaryotic subfamilies, homology between different subfamilies is very weak . Other than the three heme-binding residues (Cys13, Cys14, His18, in numeration of horse cytochrome c) there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94 and Tyr/Trp/Phe97 . In all 17 cytochromes c with known 3D-structures, these residues form a network of conserved contacts (6-94, 6-97, 10-94, 10-97 and 94-97) . Especially strong is the contact between aromatic groups in positions 10 and 97, which corresponds to 13 interatomic contacts . As residues 6, 10 and residues 94, 97 are in (i, i+4) and (i, i+3) positions in the N and C-terminal helices, respectively, the above mentioned system of conserved contacts consists mainly of contacts between one turn of N-terminal helix and one turn of C-terminal helix . The importance of the contacts between interfaces of these helices has been confirmed by the existence of these contacts in both equilibrium and kinetic molten globule-like folding intermediates, as well as by mutational evidence that these contacts are involved in tight packing between the N and C-helices . Since these four residues are not involved in heme binding and have no other apparent functional role, their conservation in highly diverged cytochromes c suggests that they are of a critical importance for protein folding . The author assumes that they are involved in a common folding nucleus of all subfamilies of c-type cytochromes .

J Biol Chem, 1998 May 15, 273(20), 12017 - 23
Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine; Zhou JJ et al.; A full-length cDNA for a membrane transporter was isolated from Brassica napus by its sequence homology to a previously cloned Arabidopsis low affinity nitrate transporter . The cDNA encodes a predicted protein of 589 amino acid residues with 12 putative transmembrane domains . The transporter belongs to a multigene family with members that have been identified in bacteria, fungi, plants, and animals and that are able to transport a range of different nitrogen-containing substrates, including amino acids, peptides, and nitrate . To identify the substrates of this plant gene, we have expressed the protein in Xenopus oocytes . The properties of the transporter are consistent with a proton cotransport mechanism for nitrate, and the voltage dependence of the Km for nitrate was determined . The Km for nitrate was shown to increase from 4 to 14 mM as the membrane voltage became more negative from -40 to -180 mV . Oocytes expressing the gene could accumulate internal nitrate to concentrations higher than those measured in water-injected controls . A range of different substrate molecules for the transporter was tested, but of these, histidine gave the largest currents, although the affinity was in the millimolar range . The pH dependence of the activity of the transporter was different for the substrates, with histidine transport favored at alkaline and nitrate at acid external pH . Kinetic analysis of the mechanism of histidine transport suggests a cotransport of protons and the neutral form of the amino acid, with the Km for histidine decreasing at more negative membrane voltages . This gene is the first member of this family of transporters for which the transport of two very different types of substrate, nitrate and histidine, has been demonstrated.

EMBO J, 1998 May 1, 17(9), 2463 - 71
Crystal structure of human uroporphyrinogen decarboxylase; Whitby FG et al.; Uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate . This activity is essential in all organisms, and subnormal activity of URO-D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT) . We have determined the crystal structure of recombinant human URO-D at 1.60 A resolution . The 40.8 kDa protein is comprised of a single domain containing a (beta/alpha)8-barrel with a deep active site cleft formed by loops at the C-terminal ends of the barrel strands . Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis . URO-D is a dimer in solution (Kd = 0.1 microM), and this dimer also appears to be formed in the crystal . Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.

Pflugers Arch, 1998 Jun, 436(1), 40 - 8
ATP-induced Ca2+ signals in bronchial epithelial cells; Sienaert I et al.; Ca2+-dependent Cl- secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released . The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o- . Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release . The expression pattern of the three InsP3 receptor isoforms was assessed both at the mRNA and at the protein level . The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level . The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed {Ca2+} rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP3 receptors . Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP3-induced Ca2+ release in permeabilized 16HBE14o- cells . We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP3 receptor and between amino acids 2030 and 2050 in the type-3 InsP3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria . We conclude that the InsP3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.

Nucleic Acids Res, 1998 May 1, 26(9), 2252 - 3
A recombination based method to rapidly assess specificity of two-hybrid clones in yeast; Petermann R et al.; The yeast two-hybrid system is frequently used to identify protein-protein interactions . Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast . In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector . Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids . Further characterization of inserts does not require plasmid isolation and intermediate hosts.

J Am Coll Surg, 1998 Jun, 186(6), 659 - 63
Density of Helicobacter pylori infection in patients with peptic ulcer perforation; Tokunaga Y et al.; BACKGROUND: A lack of change in prevalence of severe ulcer complications requiring emergency operation has been reported, despite the common use of histamine-2 (H2)-receptor antagonists and proton pump inhibitors . This may be attributable to use of ulcerogenic drugs or Helicobacter pylori (HP) infection, or both . In this study, HP infection was evaluated semiquantitatively in patients with peptic ulcer who required surgery, and the severity of histologic change was investigated . METHODS: We reviewed a total of 113 consecutive patients (98 men and 15 women) operated on for perforation, hemorrhage, or stenosis of gastroduodenal ulcer between January 1986 and December 1995 . Detection of HP was carried out by immunohistochemical staining . We graded the density of HP infection according to the number of individual HP bacteria counted in a highly magnified visual field (x 1,000 of light microscopy) . The grade of HP infection was defined as follows: (0) = 0; (1+) = 1-9; (2+) = 10-29; (3+) = 30-99; (4+) > or = 100 . The severity of gastritis was evaluated by histologic examination using the criteria of Rauws . RESULTS: Although the number of operations for gastroduodenal ulcer declined significantly, the rate of emergency operation for gastroduodenal ulcer increased from 60% to 90%, with the result that the frequency of operations for perforation or bleeding remained virtually constant and that for stenosis significantly decreased . HP infection was more prevalent in perforated ulcer (92%) than hemorrhagic ulcer (55%) or stenotic ulcer (45%) . The grades of HP infection were 3.0 +/- 0.14 (mean +/- SEM) in perforated ulcer, 2.3 +/- 0.34 in hemorrhagic ulcer, and 2.5 +/- 0.22 in stenotic ulcer . Perforated ulcer was associated with significantly more severe HP infection and gastritis changes than hemorrhagic ulcer or stenotic ulcer . CONCLUSIONS: This study indicates that patients with perforated ulcer were infected with HP more severely than those with hemorrhagic ulcer or stenotic ulcer at the time of surgery . A close relationship was observed between the perforated ulcer and the density of HP infection determined semiquantitatively using immunohistochemical stain.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 57 - 63
Serum resistance in bvg-regulated mutants of Bordetella pertussis; Fernandez RC et al.; Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance . Here we examined whether other bvg-regulated proteins contribute to serum resistance . We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type . Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants . Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2-5-fold more resistant to serum killing.

Curr Opin Struct Biol, 1998 Apr, 8(2), 150 - 8
Filamentous phage structure, infection and assembly; Marvin DA; The structural model of filamentous phage derived by X-ray fibre diffraction is supported by spectroscopic and genetic experiments . The structure of the receptor-binding domain at the end of the phage and the structure of the phage-coded intracellular DNA-binding protein have been determined at high resolution . The recent dissection of the virus life cycle by genetic and biochemical analyses, combined with structural information, suggests models for virus infection and assembly.

Nat Biotechnol, 1996 Apr, 14(4), 485 - 90
Radioactive labeling of recombinant antibody fragments by phosphorylation using human casein kinase II and {gamma-32P}-ATP; Neri D et al.; A wide range of antibody fragments can be expressed in bacteria and detected immunochemically via peptide tags . Using specially designed tags, we have developed a strategy for radiolabeling antibody fragments secreted from bacteria . Tagged antibody fragments were secreted either into the bacterial periplasm or the culture medium . The tag was not subject to proteolysis either in the broth or in human plasma . After affinity purification the antibody fragments were phosphorylated with {gamma-32P}ATP and casein kinase II . The labeled fragments were used in a gel band-shift assay to measure antigen binding affinities . In contrast to non site-specific methods such as radioiodination, antibodies labeled with casein kinase II retain full immunoreactivity . Radioactively phosphorylated antibody fragments may have many other applications, including radioimmunoassays and radioimmunotherapy.

Biochim Biophys Acta, 1998 May 27, 1364(3), 326 - 36
Action of the allelochemical, fischerellin A, on photosystem II; Srivastava A et al.; The cyanobacterium, Fischerella muscicola, produces a secondary metabolite named fischerellin A (FS) that strongly inhibits the growth of cyanobacteria and other photosynthetic organisms . The compound exhibits a unique structure and is composed of two cyclic amines and a C15 substituent that contains a double bond in the (Z) configuration and two triple bonds {L . Hagmann, F . Juttner, Tetrahedron Lett., 37 (1996) 6539-6542} . The site of FS action is located in photosystem II (PSII) . The chlorophyll fluorescence induction transient and O2 evolution methods have been used to determine the site of action of FS in PSII . FS affects the fluorescence transients, as well as O2 evolution by the cyanobacterium, Anabaena P9 . The green alga, Chlamydomonas reinhardtii, and higher plants were also affected by FS in a concentration- and time-dependent fashion . FS acts at several sites which appear with increasing half-time of interaction in the following sequence: (1) effect on the rate constant of QA- reoxidation; (2) primary photochemistry trapping; (3) inactivation of PSII reaction center; and (4) segregation of individual units from grouped units . FS does not affect the photosynthetic activity of purple bacteria, Rhodospirillum rubrum .

Biochim Biophys Acta, 1998 May 27, 1364(3), 301 - 6
A pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum; Baltscheffsky M et al.; The integrally membrane-bound, proton-pumping inorganic pyrophosphate (PPi) synthase in phototrophic bacteria is hitherto the only described alternative to the ATP synthase in biological electron transport phosphorylation . We have identified and sequenced the first gene coding for a pyrophosphate synthase . The deduced protein contains 660 amino acid residues and 15 putative membrane-spanning segments . It is homologous to the vacuolar pyrophosphatases from plants .

Rev Saude Publica, 1997 Dec, 31(6), 586 - 93
{Oral hygiene habits among Brazilian adults in an urban area of southern Brazil}; Abegg C; AIMS: This study sought to analyse the oral hygiene habits (toothbrushing frequency, use of toothpick and dental floss), of a group of Brazilian adults, in relation to socio-demographic variables . The level of dental plaque and number of teeth with gums bleeding after probing were also investigated . METHODOLOGY: The sample was composed of 234 women and 237 men, from two socioeconomic status . The age range was from 24 to 44 years . Data was collected through structured interviews and clinical examinations . RESULTS: Daily toothbrushing was frequent . The median and mode were three, and it was associated with sex and socio-economic status . The majority of the sample population (67.5%), reported using dental floss and its use was associated with sex and socio-economic status . The use of toothpicks was frequent: 54.6% of the study group used them, and their use was also associated with sex, age and social class . The majority of the sample population had a moderate level of dental plaque (62.6%) . The level of dental plaque was associated with social class . A quarter of the subjects did not have teeth with gums bleeding after probing . Bleeding gums were associated with age and social class . CONCLUSION: Oral hygiene habits were considered good for most of the participants of the study . However, improvements, are necessary among men and members of low social class.

J Inorg Biochem, 1998 Feb 15, 69(3), 203 - 7
Environmental degradation pathways for the breakdown of polydimethylsiloxanes; Stevens C; The following paper describes how polydimethylsiloxane (PDMS) polymers, which are present in a wide range of consumer products may undergo environmental degradation in the environment . The mechanism involves a biological degradation by bacteria and/or fungi, which takes place after an initial abiotic reaction initiates depolymerisation . The available information indicates that ultimately PDMS is converted to its inorganic constituents, namely carbon dioxide and silicic acid.

Ann N Y Acad Sci, 1998 May 1, 840, 359 - 72
Stress-induced enhancement of cell-mediated immunity; Dhabhar FS; We have demonstrated that acute stress induces a large-magnitude, rapid, and reversible redistribution of leukocytes from the blood to other compartments within the body . These changes in leukocyte distribution are mediated by adrenal stress hormones . Because the skin is one of the target organs of a stress-induced redistribution of leukocyes, we hypothesized that such a leukocyte redistribution could be one of the factors by which acute stress may enhance cutaneous immune function . This hypothesis was tested by examining the effects of acute stress on cutaneous delayed-type hypersensitivity (DTH) . DTH reactions are antigen-specific, cell-mediated immune responses that, depending on the antigen involved, mediate beneficial (resistance to viruses, bacteria, and fungi) or harmful (allergic dermatitis, autoimmunity) aspects of immune function . DTH was induced by challenging the pinnae of previously sensitized rats with 2,4-dinitro-1-fluorobenzene (DNFB) . Experiments showed that acute stress administered immediately before the introduction of an antigenic challenge significantly enhances a cutaneous DTH response . In contrast, chronic stress suppresses cutaneous DTH . These results demonstrate a bidirectional relationship between stress and immune function, such that acute stress enhances, while chronic stress suppresses, an important class of immune responses in vivo . They also suggest that stress-induced alterations in lymphocyte redeployment within the body may play an important role in mediating these bidirectional effects of stress on cell-mediated immunity.

Gene, 1998 May 12, 211(2), 311 - 21
The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene; Perez-Redondo R et al.; Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cephamycin and clavulanic acid biosynthesis gene cluster . claR encoded a protein of 431 amino acids (deduced Mr 47080), that showed a significant degree of homology with several transcriptional activators of the LysR family . The ClaR protein contained two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions . The second gene, car, encoded a protein of 247 amino acids (Mr 26629) that showed a strong similarity to oxydoreductases of the SDR family . Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S . clavuligerus . Amplification of the claR gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas cephamycin production was significantly reduced both in defined and in complex media . By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or cephamycin production . Both claR and car are expressed as monocistronic transcripts; the level of transcript declined rapidly after 48h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96h . claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls positively clavulanic acid and cephamycin biosynthesis . These results indicate that clavulanic acid and cephamycin biosynthesis in S . clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.

Nat Struct Biol, 1998 Jun, 5(6), 451 - 8
Tubulin and FtsZ form a distinct family of GTPases; Nogales E et al.; Tubulin and FtsZ share a common fold of two domains connected by a central helix . Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments . Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different . The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase . Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.

Nat Struct Biol, 1998 Jun, 5(6), 436 - 41
The structure of PurR mutant L54M shows an alternative route to DNA kinking; Arvidson DN et al.; The crystal structure of the purine repressor mutant L54M bound to hypoxanthine and to the purF operator provides a stereochemical understanding of the high DNA affinity of this hinge helix mutant . Comparison of the PurR L54M-DNA complex to that of the wild type PurR-DNA complex reveals that these purine repressors bind and kink DNA similarly despite significant differences in their minor groove contacts and routes to interdigitation of the central C.G:G.C base pair step . Modeling studies, supported by genetic and biochemical data, show that the stereochemistry of the backbone atoms of the abutting hinge helices combined with the rigidity of the kinked base pair step constrain the interdigitating residue to leucine or methionine for the LacI/GalR family of transcription regulators.

Biol Chem, 1998 Apr-May, 379(4-5), 585 - 9
Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I; Weiserova M et al.; We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I . It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease . While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype . The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation . This mutant resulted from a single amino acid change Trp212-->Arg . The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.

Biol Chem, 1998 Apr-May, 379(4-5), 505 - 9
Expression and characterisation of the N-terminal fragment of the HsdS subunit of M.EcoR124I; Smith MA et al.; The type IC modification methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two copies of the modification subunit, HsdM, and a single DNA specificity subunit, HsdS . Studies to date have been largely restricted to the HsdM subunit or the intact methyltransferase, since the HsdS subunit is insoluble when expressed independently of HsdM . Using PCR, we have cloned and expressed 13 fragments of the gene for the HsdS subunit, including the sequences encoding each of the variable and conserved domains and various combinations of these . Only two of these fragments were found to be soluble, a 8.6 kDa fragment (S11) comprising the central conserved domain and a 25 kDa N-terminal fragment (S3) containing the N-terminal variable domain and the central conserved domain . Analysis of the larger of these fragments by gel retardation shows that the protein binds DNA in the presence of HsdM at a subunit stoichiometry of 1:1 . Gel filtration and CD spectroscopy indicate that the protein is monomeric and predominantly alpha-helical.

J Eukaryot Microbiol, 1998 May-Jun, 45(3), 293 - 7
Monophyly of endosymbiont containing trypanosomatids: phylogeny versus taxonomy; Hollar L et al.; To obtain additional information on the phylogenetic relationships within the family Trypanosomatidae (order Kinetoplastida), we have sequenced the small subunit ribosomal RNA genes from the endosymbiont containing species Herpetomonas roitmani TCC080, Herpetomonas sp . TCC263, Crithidia oncopelti ATCC 12982 and a partial large subunit rRNA gene from H . roitmani . The small subunit sequences in the two isolates of Herpetomonas are very similar but not identical, and so are their restriction digest profiles of kinetoplast DNA . The size of minicircles in both isolates is 4.2 kilobases . The inferred ribosomal RNA phylogenetic trees shows the genera Herpetomonas and Crithidia as polyphyletic . Endosymbiont-bearing herpetomonads cluster with the endosymbiont-bearing crithidias and a blastocrithidia to form a monophyletic clade, whereas the endosymbiont-free members of these genera are found elsewhere in the tree . These data support the hypothesis of a monophyletic origin of endosymbiosis in trypanosomatid evolution and also suggest that a taxonomic revision is needed in order to better describe the natural affinities in this family.

FEMS Immunol Med Microbiol, 1998 Apr, 20(4), 275 - 81
Inhibition of Helicobacter pylori sialic acid-specific haemagglutination by human gastrointestinal mucins and milk glycoproteins; Hirmo S et al.; Helicobacter pylori, a human gastric pathogen causing chronic gastritis and duodenal ulcer disease, has been found in large amounts in gastric mucous gel layer . Mucin preparations, separated from human gastric juices and isolated from different colon regions, were examined for their ability to inhibit haemagglutination of H . pylori with the emphasis on evaluating the role of sialic acid-dependent haemagglutinins of the bacteria in colonisation of the stomach . The mucins showed high inhibitory activity for H . pylori, which was significantly decreased after the removal of sialic acids from the mucins . The inhibitory potencies using high molecular mass mucin-like components from bovine milk were comparable with those obtained for gastric mucins, suggesting their possible role in the prevention of H . pylori infection.

Free Radic Biol Med, 1998 May, 24(7-8), 1120 - 9
Procyanidins extracted from Pinus maritima (Pycnogenol): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages; Virgili F et al.; Nitrogen monoxide (NO) has diverse physiological roles and also contributes to the immune defense against viruses, bacteria, and other parasites . However, excess production of NO is associated with various diseases such arthritis, diabetes, stroke, septic shock, autoimmune, chronic inflammatory diseases, and atheriosclerosis . Cells respond to activating or depressing stimuli by enhancing or inhibiting the expression of the enzymatic machinery that produce NO . Thus, maintenance of a tight regulation of NO production is important for human health . Phytochemicals have been traditionally utilized in ways to treat a family of pathologies that have in common the disregulation of NO production . Here we report the scavenging activity of Pycnogenol (the polyphenols containing extract of the bark from Pinus maritima) against reactive oxygen and nitrogen species, and its effects on NO metabolism in the murine macrophages cell line RAW 264.7 . Macrophages were activated by the bacterial wall components lipopolysaccharide (LPS) and interferon (IFN-gamma), which induces the expression of large amounts of the enzyme nitric oxide synthase (iNOS) . Preincubation of cells with physiological concentrations of Pycnogenol significantly decreased NO generation . It was found that this effect was due to the combination of several different biological activities, i.e., its ROS and NO scavenging activity, inhibition of iNOS activity, and inhibition of iNOS-mRNA expression . These data begin to provide the basis for the conceptual understanding of the biological activity of Pycnogenol and possibly other polyphenolic compounds as therapeutic agents in various human disorders.

Biotechnol Prog, 1998 May-Jun, 14(3), 393 - 409
Effects of oxygen on recombinant protein expression; Konz JO et al.; Efforts to increase cell growth and protein yields need to be complemented by the maintenance of the quality of the protein produced . Elevated oxygen pressure or rapid increases in oxygen content can cause oxidative stress within the cells, leading to oxidation of specific proteins and nucleotide sequences . In addition, transient or steady-state anoxic conditions can cause limitations in amino acid production and plasmid stability . Major pathways and mechanisms of oxidative damage to proteins expressed in bacteria are reviewed . Damage to nucleic acids involved in gene expression also is considered . The methodologies for identifying oxidative damage to macromolecules are improving but are not yet adequate for on-line feedback . This limits our ability to integrate information about these phenomena and the cellular responses into a quantitative model . Enough information is available, however, to consider changes in the time profile of dissolved oxygen as a cause for poor process performance.

Am J Gastroenterol, 1998 May, 93(5), 726 - 31
Analysis of risk factors for chronic hepatic encephalopathy: the role of Helicobacter pylori infection; Dasani BM et al.; OBJECTIVE: Elevated blood ammonia is an important pathogenic factor of hepatic encephalopathy . Although colonic bacteria are considered the main source of ammonia, the stomach in subjects with urease-producing Helicobacter pylori (H . pylori) is an alternative site . The objective of this study was to determine whether H . pylori is associated with this complication . METHODS: After assessing liver function and portal hypertension, 55 cirrhotics were evaluated for encephalopathy and H . pylori infection . Response to 2 weeks of amoxicillin (2 g/day) and omeprazole (40 mg/day) was then assessed in 17 (13 H . pylori-positive, four H . pylori-negative) encephalopathic subjects . RESULTS: H . pylori infection was more common (67 % vs 33%, p = 0.004) among encephalopathic patients . Additional factors associated with encephalopathy included older age (60.1 +/- 1.5 vs 49.8 +/- 2.4 yr, p = 0.001), lower albumin (3.17 +/- 0.08 vs 3.69 +/- 0.12 g/dl, p = 0.001), higher total bilirubin (2.24 +/- 0.20 vs 1.53 +/- 0.23 mg/dl, p = 0.034), greater ascites score (0.8 +/- 0.1 vs 0.3 +/- 0.1, p = 0.01), greater diuretic score (1.1 +/- 0.1 vs 0.3 +/- 0.1, p = 0.002), and greater modified Child score (6.7 +/- 0.3 vs 5.1 +/- 0.3, p = 0.001) . When adjusted for severity of cirrhosis and age, H . pylori continued to demonstrate a statistical association (p = 0.039) . After anti-H . pylori therapy, symptomatology in infected encephalopathic patients appeared to improve, whereas noninfected subjects were unaffected . CONCLUSION: In cirrhotic patients, H . pylori infection is associated with hepatic encephalopathy, especially in younger patients with decompensated liver disease.

J Exp Med, 1998 May 18, 187(10), 1659 - 69
The effect of class II major histocompatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage; Fan X et al.; Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer . Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis . Using H . pylori as "bait" to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor . Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules . Moreover, binding of H . pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC . Since type 1 T helper cells are present during infection and produce interferon (IFN)-gamma, which increases class II MHC expression, gastric epithelial cell lines were exposed to H . pylori in the presence or absence of IFN-gamma . IFN-gamma increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells . In contrast to MHC II-negative cell lines, H . pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection . These data describe a novel receptor for H . pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.

J Clin Invest, 1998 May 1, 101(9), 1932 - 41
The agent of Human Granulocytic Ehrlichiosis resides in an endosomal compartment; Webster P et al.; The composition of cytoplasmic vacuoles containing the agent of Human Granulocytic Ehrlichiosis (HGE) was studied to investigate how this pathogen exists within infected host cells . Electron microscopy demonstrated that the HGE organism resides in a membrane-bound compartment within HL-60 cells: early forms of the HGE agent have a round reticular appearance while later structures are small and dense . Vacuoles containing HGE bacteria incorporated endocytosed colloidal gold particles, suggesting that they are part of the endocytic pathway . Antibodies directed to the mannose-6-phosphate receptor labeled vacuole membranes . Antibodies to the transferrin receptor and to the lysosomal membrane glycoprotein LAMP 1 did not . Moreover, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, which normally accumulates in compartments with low pH, was not present inside these vacuoles . These results suggest that vacuoles containing the agent of HGE fail to mature into phagolysosomes . We conclude that the agent of HGE appears to enter and modify part of the endocytic pathway.

J Nutr, 1998 May, 128(5), 843 - 7
Stimulation of epithelial cell proliferation of isolated distal colon of rats by continuous colonic infusion of ammonia or short-chain fatty acids is nonadditive; Ichikawa H et al.; Dietary fibers accelerate colonic epithelial cell proliferation at least in part by modulating bacterial metabolism in the large intestine . Ammonia and short-chain fatty acids (SCFA) are major metabolites of hindgut bacteria and are believed to affect epithelial cell kinetics of the colon . However, the effect of luminal ammonia itself and the possible interaction of ammonia with SCFA on colonic epithelial cell proliferation have not yet been studied . The colon of rats was surgically isolated and continuously administered infusates with saline, ammonia, SCFA or both into the isolated colon for 7 d in a two-way factorial design . On d 7, vincrystine sulfate was administered intravenously to cause metaphase arrest . The activity of epithelial cell proliferation in the distal colon was estimated by using a stathmokinetic method and by histologic examination . The crypt size was significantly larger in rats given infusates containing SCFA than in rats given infusates without SCFA . Infusion of ammonia or SCFA significantly stimulated colonic epithelial cell proliferation compared with the saline infusion . Infusion of both ammonia and SCFA resulted in accumulated mitoses per crypt that did not differ from the other three infusions although the value tended to be lower than when SCFA alone were infused . Thus, stimulation of epithelial cell proliferation by ammonia and SCFA is not additive, and the interaction between them should be considered when the effects of dietary fibers on gut epithelial proliferation are investigated.

Plant Cell, 1998 Apr, 10(4), 599 - 612
A glycoprotein modified with terminal N-acetylglucosamine and localized at the nuclear rim shows sequence similarity to aldose-1-epimerases; Heese-Peck A et al.; Several glycoproteins that are present at the nuclear rim and at the nuclear pore complex of tobacco suspension-cultured cells are modified by O-linked oligosaccharides with terminal N-acetylglucosamine (GlcNAc) . Here, we report on the purification of several of these glycoproteins, which are referred to as terminal GlcNAc (tGlcNAc) proteins . In vitro galactosylation of the tGlcNAc proteins generated glycoproteins with terminal galactosyl-beta-1, 4-GlcNAc and thus permitted their isolation by Erythrina crystagalli agglutinin affinity chromatography . Peptide sequence information derived from one tGlcNAc protein with an apparent molecular mass of 40 to 43 kD, designated gp40, made it possible to clone its gene . Interestingly, gp40 has 28 to 34% amino acid identity to aldose-1-epimerases from bacteria, and no gene encoding an aldose-1-epimerase has been isolated previously from higher organisms . Polyclonal antibodies were generated against recombinant gp40 . Consistent with its purification as a putative nuclear pore complex protein, gp40 was localized to the nuclear rim, as shown by biochemical fractionation and immunofluorescence microscopy.

J Bioenerg Biomembr, 1998 Feb, 30(1), 25 - 33
Regulation of energy transduction and electron transfer in cytochrome c oxidase by adenine nucleotides; Kadenbach B et al.; Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP . One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H+/e- stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP . Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K(M) for cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively . We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H+/e- stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals . This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.

Transfus Clin Biol, 1998 Apr, 5 Suppl 1, 9S - 32S
{Blood groups and structure-activity relations}; Cartron JP; In the recent years, advances in biochemistry and molecular genetics have contributed to establish the structure of the genes and proteins from most of the 23 blood group systems presently known . From these findings, five functional classes of molecules can be schematically distinguished: (i) transporters and channels, (ii) receptors for ligands, viruses, bacteria and parasites, (iii) adhesion molecules, (iv) enzymes, and (v) structural proteins . Recent advances on these molecules will be reviewed, particularly by illustrating available structure-function relationships.

Mol Microbiol, 1998 Apr, 28(2), 281 - 91
Specific RecA amino acid changes affect RecA-UmuD'C interaction; Sommer S et al.; The UmuD'C mutagenesis complex accumulates slowly and parsimoniously after a 12 Jm(-2) UV flash to attain after 45 min a low cell concentration between 15 and 60 complexes . Meanwhile, RecA monomers go up to 72,000 monomers . By contrast, when the UmuD'C complex is constitutively produced at a high concentration, it inhibits recombinational repair and then markedly reduces bacterial survival from DNA damage . We have isolated novel recA mutations that enable RecA to resist UmuD'C recombination inhibition . The mutations, named recA {UmuR}, are located on the RecA three-dimensional structure at three sites: (i) the RecA monomer tail domain (four amino acid changes); (ii) the RecA monomer head domain (one amino acid change, which appears to interface with the amino acids in the tail domain); and (iii) in the core of a RecA monomer (one amino acid change) . RecA {UmuR} proteins make recombination more efficient in the presence of UmuD'C while SOS mutagenesis is inhibited . The UmuR amino acid changes are located at a head-tail joint between RecA monomers and some are free to possibly interact with UmuD'C at the tip of a RecA polymer . These two RecA structures may constitute possible sites to which the UmuD'C complex might bind, hampering homologous recombination and favouring SOS mutagenesis.

Appl Biochem Biotechnol, 1998 Apr, 73(1), 19 - 28
Conformational stability and antibody response to the 18kDa heat-shock protein formulated into different vehicles; Costa MH et al.; Protein stability is one of the most important obstacles for successful formulation in the development of new-generation vaccines . Here, the 18kDa heat-shock protein (18kDa-hsp) was chemically modified though conjugation with bovine serum albumin or by esterification with N-hydroxysuccinimide ester of palmitic acid . The biologically active conformation of the protein was preserved after chemical modification . The immune responses to the recombinant 18kDa-hsp from Mycobacterium leprae were studied in different presentations: free, copolymerized with bovine serum albumin in aggregates (18kDa-hsp-BSA), and either surface linked to liposomes or entrapped into liposomes . Measuring the antibody production of immunized genetically selected mice has compared the adjuvant effects of liposomes and proteic copolymer . Among the two liposome preparations, the strongest response was obtained with the surface-exposed antigen-liposomes . The copolymer 18kDa-hsp-BSA conferred a high titer of antibody in injected mice, and persisted 70 d after immunization . This approach should prove very useful for designing more effective vaccines by using 18kDa-hsp as carrier protein.

J Bacteriol, 1998 Jun, 180(12), 3253 - 6
The FixK2 protein is involved in regulation of symbiotic hydrogenase expression in Bradyrhizobium japonicum; Durmowicz MC et al.; The roles of the nitrogen fixation regulatory proteins NifA, FixK1, and FixK2 in the symbiotic regulation of hydrogenase structural gene expression in Bradyrhizobium japonicum have been investigated . Bacteroids from FixJ and FixK2 mutants have little or no hydrogenase activity, and extracts from these mutant bacteroids contain no hydrogenase protein . Bacteroids from a FixK1 mutant exhibit wild-type levels of hydrogenase activity . In beta-galactosidase transcriptional assays with NifA and FixK2 expression plasmids, the FixK2 protein induces transcription from the hup promoter to levels similar to those induced by HoxA, the transcriptional activator of free-living hydrogenase expression . The NifA protein does not activate transcription at the hydrogenase promoter . Therefore, FixK2 is involved in the transcriptional activation of symbiotic hydrogenase expression . By using beta-galactosidase transcriptional fusion constructs containing successive truncations of the hup promoter, the region of the hup promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site.

Eur J Med Res, 1998 Jun 17, 3(6), 295 - 8
Fructose malabsorption is associated with early signs of mental depression; Ledochowski M et al.; Fructose malabsorption is characterized by the inability to absorb fructose efficiently . As a consequence fructose reaches the colon were it is broken down by bacteria to short fatty acids, CO2 and H2 . Bloating, cramps, osmotic diarrhea and other symptoms of irritable bowel syndrome are the consequence and can be seen in about 50% of fructose malabsorbers . Having made the observation that persons with fructose malabsorption very often seem to present not only with signs of irritable bowel syndrome but also with signs of pre-menstrual syndrome and mental depression, it was of interest to establish whether such an association could be demonstrated in patients . Fifty-five adults with gastrointestinal complaints of unknown origin (12 males, 43 females) were analyzed by measuring breath hydrogen concentrations after an oral dose of 50 g fructose and were classified as normals or fructose malabsorbers according to their breath H2 concentrations . All patients filled out a Beck s depression inventory - questionnaire . Fructose malabsorption was detected in 36 of 55 individuals (65.5%) . Subjects with fructose malabsorption (DeltaH2 concentrations >10 p.p.m . after fructose load) showed a significantly higher score in the Beck s depression inventory than normal fructose absorbers . This was true especially for females . Fructose malabsorption may play a role in the development of depressed mood . Fructose malabsorption should be considered in patients with symptoms of major depression or pre-menstrual syndrome . Further studies are needed to clarify the background of this association.

Cytokine Growth Factor Rev, 1997 Dec, 8(4), 293 - 312
The growing family of interferon regulatory factors; Nguyen H et al.; Interferons (IFN) exert their multiple biological effects through the induction of expression of over 30 genes encoding proteins with antiviral, antiproliferative and immunomodulatory functions . Among the many IFN-inducible proteins are the Interferon Regulatory Factors (IRFs), a family of transcription regulators, originally consisting of the well-characterized IRF-1 and IRF-2 proteins; the family has now expanded to over 10 members and is still growing . The present review provides a detailed description of recently characterized IRF family members . Studies analyzing IRF-expressing cell lines and IRF knockout mice reveal that each member of the IRF family exerts distinct roles in biological processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development . Understanding the molecular mechanisms by which the IRFs affect these important cellular events and IFN expression will contribute to a greater understanding of events leading to various viral, immune and malignant disease states and will suggest novel strategies for antiviral and immune modulatory therapy.

Toxicon, 1998 Feb, 36(2), 247 - 55
First report of microcystins in Taiwan; Lee TH et al.; This is the first report on microcystins from Microcystis aeruginosa Kutzing in Taiwan . A total of nine strains of cyanobacteria have been isolated from eutrophic aquaculture ponds and water reservoirs . By mouse toxicity assay, six of the nine strains had LD100 in the range of 25-100 mg per kg mouse for dried bacterial mass . Microcystin-LR and -RR were found in all toxic strains and their contents ranged from 0.11-10.06 microg and 0.08 2.21 microg per mg of dried bacteria, respectively . Microcystin-RA, a minor component found only in M . TN-2 and M . CY-1 strains, was identified as a new microcystin . All three toxins were isolated by a serial separation on an LH-20 column, Si-flash column chromatography and reverse phase HPLC . Toxins were further identified by comparing their FABMS, 1H and 1H-1H COSY NMR spectra with the authentic microcystin-LR . Several other microcystin-like compounds were also found in the cultured strains and their structures are being determined.

Int J Dermatol, 1998 May, 37(5), 370 - 7
Community health workers reduce skin diseases in East African children; Schmeller W; BACKGROUND: Epidemiologic data concerning skin diseases in many rural areas in sub-Saharan Africa are not available . Little is known about the effect of regular treatment schedules by paramedical staff (especially community health workers) in the primary healthcare system on the severity and prevalence of dermatoses . METHODS: 5780 school and pre-school children from 13 primary schools in four sublocations in rural western Kenya (Kisumu District) were examined for dermatoses by the author, together with community health workers in 1993 . On-the-spot training and weekend seminars about important and common dermatoses were also given . In 1994 a dermatology program was started within the primary healthcare system . Twelve trained community health workers carried out regular school visits once a week and diagnosed and treated pupils with dermatoses . Treatment was performed with gentian violet 1% solution for bacterial skin infections, Whitfield's ointment for dermatophytoses, benzylbenzoate emulsion 25% for scabies, and hydrocortisone acetate 1% cream for eczemas . All schools were visited again in 1995 to evaluate the long-term effects of the program . RESULTS: In 1993, the prevalence rate for dermatoses was 32.4% . Most of the skin diseases found were of infective origin (27.1% were caused by bacteria, 21.6% by fungi, and 17.6% by arthropods, mainly scabies mites) . Dermatitis accounted for 3.5% . In 1995, the prevalence of dermatoses declined to 29.6% (p<0.05), and this reduction was most strongly observed for tropical ulcers and tinea capitis . Additionally, there was an improvement in the extent and severity of skin diseases . CONCLUSIONS: This study defines, for the first time, the number and extent of skin diseases in children in rural Kisumu District; most dermatoses were of infective origin . The study demonstrates that community health workers in the primary healthcare system are capable of dealing successfully with the most common dermatoses in children following a short training period.

J Clin Microbiol, 1998 Jun, 36(6), 1501 - 11
Production and characterization of Ehrlichia risticii, the agent of Potomac horse fever, from snails (Pleuroceridae: Juga spp.) in aquarium culture and genetic comparison to equine strains; Reubel GH et al.; We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains . Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory . Upon exposure to temperatures above 22 degrees C the snails released trematode cercariae tentatively identified as virgulate cercariae . Fragments of three different genes (genes for 16S rRNA, the groESL heat shock operon, and the 51-kDa major antigen) were amplified from cercaria lysates by PCR and sequenced . Genetic information was also obtained from E . risticii strains from horses with PHF . The PCR positivity of snail secretions was associated with the presence of trematode cercariae . Sequence analysis of the three genes indicated that the source organism closely resembled E . risticii, and the sequences of all three genes were virtually identical to those of the genes of an equine E . risticii strain from a property near the snail collection site . Phylogenetic analyses of the three genes indicated the presence of geographical E . risticii strain clusters.

Planta Med, 1998 May, 64(4), 295 - 302
Biologically active substances from the genus Artemisia; Tan RX et al.; Artemisia species, widespread in nature, are frequently utilized for the treatment of diseases such as malaria, hepatitis, cancer, inflammation, and infections by fungi, bacteria, and viruses . Furthermore, some Artemisia constituents were found to be potential insecticides and allelopathic chemicals . This genus is receiving growing attention presumably due to: (i) the diversified biology and chemistry of the constituents, (ii) the frequent application in traditional medical practice, and (iii) the rich source of the plant material . This review summarizes mainly the biological results obtained in the past decade . The significance and trends in this field are briefly discussed.

Dig Dis, 1998 May-Jun, 16(3), 159 - 68
Gastric stump cancer: what is the risk?
Safatle-Ribeiro AV, Ribeiro U Jr, Reynolds JC.
Patients who have undergone partial gastric resections are at an increased risk for the development of cancer in the gastric remnant . The overall risk increases over time and is higher in patients with an initial diagnosis of gastric rather than duodenal ulcer, in men and following partial gastrectomy with Billroth II reconstruction . The site of tumor growth is predominantly in the anastomotic area, but may occur anywhere in the stump . Enterogastric reflux, achlorhydria, bacteria overgrowth, and Helicobacter pylori appear to be the major factors involved in the etiopathogenesis of the gastric stump cancer . Surveillance of these patients with endoscopy and multiple biopsies may provide the means to diagnose tumors at an early stage, but the cost-benefit ratio of surveillance requires further study . Despite the magnitude of alterations in gastric stump mucosa, unfortunately, at this time we do not have good predictors of patients who will develop a cancer.

Dev Comp Immunol, 1998 Jan-Feb, 22(1), 1 - 12
Characterization of a 14 kDa plant-related serine protease inhibitor and regulation of cytotoxic activity in earthworm coelomic fluid; Roch P et al.; We have purified and characterized the serine protease inhibitor activity contained in the coelomic fluid of the earthworms, Eisenia . Serine protease inhibitor activity was stable between pH3 and 9.5, not flocculable by pH 3.0 and resistant to 100 degrees C for 15 min . or to 4 degrees C for 24 h . Ten microL of coelomic fluid was sufficient to inhibit in vitro the protease activity of 0.12 microgram of trypsin . Injection of living bacteria into earthworms resulted in increased serine protease activity 1-2 days post-injection, and increased serine protease inhibitor activity on day 4, suggesting that serine protease inhibitor is responsible for serine protease neutralization . Purified to homogeneity by affinity chromatography on trypsin, the serine protease inhibitor of Eisenia is a monomer of 14 kDa . Its partial NH2 amino acid sequence revealed a basic hydrophobic fragment which shared 68-75% homologies and 47-60% identities with several plant serine protease inhibitors . Eisenia cytotoxic activity due to the two fetidins of 40 and 45 kDa was stimulable in vitro by several serine proteases . Incubation with soybean trypsin inhibitor variant a (STIa) resulted in less cytotoxicity . The inhibitory effect occurred only when STIa was added before cell disruption . Interpretative cytotoxic scheme involving the release of intracellular cytotoxic proteins, intracellular trypsin-like activator and extracellular serine protease inhibitor suggests regulatory mechanisms for cellular/humoral immune system of earthworms.

J Pathol, 1998 Mar, 184(3), 323 - 31
Lack of inducible nitric oxide synthase in bronchial epithelium: a possible mechanism of susceptibility to infection in cystic fibrosis; Meng QH et al.; Cystic fibrosis (CF) is an inherited disorder associated with severe inflammation and repeated bacterial infection and colonization in the lung . Airway epithelium is involved in defence against bacteria, but this system may be defective in CF . Pro-inflammatory cytokines can stimulate the expression of inducible nitric oxide synthase (iNOS), an enzyme generating nitric oxide, which functions as an important mediator in host defence mechanisms . To understand better the poor resistance to infections in the CF lung, the expression of the iNOS gene was investigated in explanted lungs from patients with cystic fibrosis (n = 13), bronchiectasis (n = 3), emphysema (n = 14), and in normal lungs (n = 8) . In addition, bronchial epithelial cell lines were examined to study iNOS gene expression in vitro . Strong immunoreactivity for iNOS was seen in inflammatory cells and bronchial epithelium in all the diseased lungs, except for bronchial epithelium in CF . Quantitative analysis showed a significant reduction in the area of epithelium immunostained in CF {CF 6.8 +/- 1.6 (% +/- SEM); emphysema 18.2 +/- 2.8; normal 9.6 +/- 0.8, P < 0.01}, regardless of steroid treatment . These results were supported by in situ hybridization of iNOS mRNA, which showed a pattern of gene expression in CF, emphysema, and normal lung which paralleled that of protein immunoreactivity . Stimulation with cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) increased the expression of iNOS mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultures of normal (16HBE14o-), but not CF (CFBE41o-, with delta F508 CFTR mutation) epithelial cells . Expression of iNOS in inflammatory cells suggests that the gene is normal in CF . Absence of iNOS from bronchial epithelium may be due to low expression of the gene resulting from abnormalities in the signalling system that normally causes induction, such as cytokine receptors, second messengers or transcription factors . The resulting deficiency of the nitric oxide defence system may be relevant to the susceptibility of CF patients to pulmonary bacterial colonization.

Plant Cell Physiol, 1998 Apr, 39(4), 411 - 7
Growth, pigmentation, and expression of the puf and puc operons in a light-responding-repressor (SPB)-disrupted Rhodobacter sphaeroides; Nishimura K et al.; We previously cloned a trans-repressor, SPB, for the puf operon of Rhodobacter sphaeroides (Shimada et al . 1996) and revealed that SPB was a putative genetic counterpart to HvrA in Rhodobacter capsulatus, a trans-activator for the puf and puh operons (Mizoguchi et al . 1997) . In this study we constructed a spb-disrupted R . sphaeroides, strain L-7, to elucidate the function of SPB . This disruption of the spb gene increased the photosynthetic growth rate and the cellular levels of photopigments under low-intensity light conditions . The disruption also derepressed the expression of the puf and puc operons under high-intensity light conditions . In strain L-7, however, strong illumination still reduced the cellular levels of photopigments as it did in the wild strain, suggesting that SPB did not directly affect the formation of photopigments . These results support our previous suggestion that SPB functions as a high-light repressor for puf operon in R . sphaeroides in striking contrast to HvrA, which is a low-light activator for puf and puh operons in R . capsulatus, even though SPB and HvrA are highly homologous . Disruption of spb gene had no effect on the oxygen-mediated regulation of the pigmentation or the expression of puf and puc operons.

Mol Biol Evol, 1998 Jun, 15(6), 683 - 9
Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin; Peyretaillade E et al.; An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia) . Southern blot analyses show the presence of a single gene copy located on chromosome XI . The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs . Phylogenetic analysis using maximum likelihood and evolutionary distances place the E . cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences . Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E . hellem . The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins . No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification . These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia . The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

Cell Mol Life Sci, 1998 Apr, 54(4), 305 - 8
Cell wall polymers in Archaea (Archaebacteria); Kandler O et al.; The distribution of the various cell wall and cell envelope (S-layer) polymers among the main lineages of the domain Archaea (Archaebacteria) and the chemical composition and primary structure of polymers forming rigid cell wall sacculi is described . Differences between bacteria and archaea in their sensitivity to antibiotics which inhibit cell wall synthesis in bacteria are discussed.

J Bacteriol, 1998 Jun, 180(11), 2911 - 4
glkA is involved in glucose repression of chitinase production in Streptomyces lividans; Saito A et al.; Chitinase production in Streptomyces lividans is induced by chitin and repressed in the presence of glucose . A mutant of S . lividans TK24, strain G015, which was defective in glucose repression of chitinase production, was obtained by screening colonies for zones of clearing on colloidal chitin agar plates containing 1.0% (wt/vol) glucose . The transcriptional analysis of chiA in G015 with xylE, which encodes catechol 2,3-dioxygenase, as a reporter gene showed that the transcription from the chiA promoter of S . lividans TK24 occurred regardless of the presence of glucose . G015 was resistant to 2-deoxyglucose (2-DOG) and did not utilize glucose as a sole carbon source . When a DNA fragment containing glkA, a gene for glucose kinase, of Streptomyces coelicolor A3(2) was introduced into strain G015 on a low-copy-number plasmid, the sensitivity to 2-DOG, the ability to utilize glucose, and the glucose repression of chitinase production were restored . These results indicate that glkA is involved in glucose repression of chitinase production in S . lividans TK24.

J Neurosci, 1998 Jun 1, 18(11), 4008 - 21
Mitogen-activated protein kinases (Erk1,2) phosphorylate Lys-Ser-Pro (KSP) repeats in neurofilament proteins NF-H and NF-M; Veeranna et al.; Mammalian neurofilament proteins, particularly midsized (NF-M) and heavy (NF-H) molecular weight neurofilament proteins, are highly phosphorylated in axons . Neurofilament function depends on the state of phosphorylation of the numerous serine/threonine residues in these proteins . Most phosphorylation occurs in the lys-ser-pro (KSP) repeats in the C-terminal tail domains of NF-H and NF-M . In our previous study, cyclin-dependent kinase 5 (cdk5) was shown to phosphorylate specifically the KSPXK repeats in rat NF-H . Because 80% of the repeats are of the KSPXXXK type, it was of interest to determine which kinase phosphorylates these motifs . Using a synthetic KSPXXXK peptide to screen for a specific kinase, we fractionated rat brain extracts by column chromatography and identified extracellular signal-regulated kinase (Erk2) activated by an upstream activator, the mitogen-activated protein kinase kinase MAPKK (MEK), by Western blot analysis, sequence identification, and inhibition by a specific MEK inhibitor (PD 98059) . The fraction containing Erk2, as well as bacterially expressed Erk1 and Erk2, phosphorylated all types of KSP motifs in peptides (KSPXK, KSPXXK, KSPXXXK, and KSPXXXXK) derived from NF-M and NF-H . They also phosphorylated an expressed 24 KSPXXXK repeat NF-H polypeptide, an expressed NF-H as well as dephosphorylated native rat NF-H, and NF-M proteins with accompanying decreases in their respective electrophoretic mobilities . A comparative kinetic study of Erk2 and cdk5 phosphorylation of KSPXK and KSPXXXK peptides revealed that, in contrast to cdk5, which phosphorylated only the KSPXK peptide, Erk2 could phosphorylate both . The preferred substrate for Erk2 was KSPXXXK peptide . The MEK inhibitor PD 98059 also inhibited phosphorylation of NF-H, NF-M, and microtubule-associated protein (MAP) in primary rat hippocampal cells and caused a decrease in neurite outgrowth, suggesting that Erk1,2 may play an important role in neurite growth and branching . These data suggest that neuronal Erk1 and Erk2 are capable of phosphorylating serine residues in diverse KSP repeat motifs in NF-M and NF-H.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5395 - 400
Cloning and characterization of a potassium-coupled amino acid transporter; Castagna M et al.; Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na+ or H+ gradients . The present work extends the Na+ and H+ dogma by including the H+ and K+ paradigm . Lepidopteran insect larvae have a high K+ and a low Na+ content, and their midgut cells lack Na+/K+ ATPase . Instead, an H+ translocating, vacuolar-type ATPase generates a voltage of approximately -240 mV across the apical plasma membrane of so-called goblet cells, which drives H+ back into the cells in exchange for K+, resulting in net K+ secretion into the lumen . The resulting inwardly directed K+ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells . By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K+-coupled amino acid transporter (KAAT1) . We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library . KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands . When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes alpha-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B . K+, Na+, and to a lesser extent Li+ were accepted as cotransported ions, but K+ is the principal cation, by far, in living caterpillars . Moreover, uptake was Cl(-)-dependent, and the K+/Na+ selectivity increased with hyperpolarization of oocytes, reflecting the increased K+/Na+ selectivity with hyperpolarization observed in midgut tissue . KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na+ and Cl(-)-coupled neurotransmitter transporter family.

Glycobiology, 1998 Apr, 8(4), 297 - 309
The lactosylceramide binding specificity of Helicobacter pylori; Angstrom J et al.; The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay . Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere . By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding . A selective binding of H . pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay . Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers . An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin . By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases . Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

FEBS Lett, 1998 May 1, 427(1), 79 - 84
Cloning and functional expression of a 'fast' fungal kinesin; Grummt M et al.; Conventional kinesins are molecular motors that move towards the plus end of microtubules . In animal species, they have been shown to be remarkably conserved in terms of both their primary sequence and several physiological properties, including their velocity of movement . Here we report the cloning of Synkin, a homologue of conventional kinesin from the zygomycete fungus Syncephalastrum racemosum {Steinberg, Eur . J . Cell Biol . 73 (1997) 124-131} that is 4-5 times faster than its animal counterparts . Expression in bacteria yields a fully functional motor that moves at the same speed as the native motor isolated from fungal hyphae and has similar hydrodynamic properties . Its sequence is most closely related to that of two other fungal kinesins from Neurospora and Ustilago, and shares several biochemical properties with the Neurospora motor . Fungal kinesins therefore seem to form a conserved subfamily of conventional kinesins distantly related to animal kinesins . They may help to identify sequence features important for determining motor velocity.

Mol Gen Genet, 1998 Apr, 258(1-2), 133 - 8
Refined genetic map of the obligate methylotroph Methylobacillus flagellatum; Serebrijski I et al.; We present a refined genetic map of the obligate methylotroph Methylobacillus flagellatum . New, Hfr (high-frequency-of-transfer) donors, and pulsed-field gel electrophoresis, were used to determine that M . flagellatum contains one approximately 3.1-Mb circular chromosome, and no plasmids . A correlation between time-of-entry units and DNA length was established . Using in vivo and in vitro cloning, and sequencing, a number of new genetic markers were identified and mapped; in addition, the nature of some of the previously mapped markers was elucidated.

Ann Rheum Dis, 1998 Feb, 57(2), 100 - 6
Analysis of T cell subsets present in the peripheral blood and synovial fluid of reactive arthritis patients; Beacock-Sharp H et al.; OBJECTIVE: Reactive arthritis (ReA), a HLA-B27 associated arthropathy, develops in susceptible people after infection with certain bacteria . T cells have been implicated in the pathogenesis of the arthritis but which of the different subsets is involved is still debated . This study has further elucidated the role of the CD4+ and CD8+ T cells by examining the expression of various surface markers associated with activation . METHODS: Three colour flow cytometry was used to examine the phenotype of the T cells within the synovial fluid (SF) and peripheral blood (PB) of ReA patients . RESULTS: ReA SF, compared with paired PB, contained a higher percentage of CD69+, CD25+, and HLA-DR+ CD3+ T cells . The majority of SF T cells also expressed the putative memory marker CD45RO . Within the T cell subsets, CD25 was expressed primarily on the CD4+ T cells; however more CD8+ T cells were HLA-DR+ . CONCLUSION: The results show that both CD4+ and CD8+ T cell populations demonstrate evidence of recent activation . Whether these cells are involved in inducing inflammation, regulating the inflammation, or have become active as a result of migration through the endothelium, remains to be determined by functional studies.

J Clin Laser Med Surg, 1997, 15(4), 185 - 8
The carbon dioxide laser as an aid in apicoectomy: an in vitro study; Moritz A et al.; OBJECTIVE: To achieve the required goal of optimally sealing the apical section and the root-canal when performing an apicoectomy, the authors decided to use the CO2 laser as an additional aid . SUMMARY BACKGROUND DATA: The CO2 laser has previously shown to have an excellent sealing effect on dentin surfaces . METHODS: In this in vitro study, the authors examined the effects of CO2 laser application in apicoectomies with the help of color penetration tests and scanning electron microscopic (SEM) examinations . Sections and root canals were irradiated with low power (0.5 W) in continuous wave mode for totally 20 sec . The thermal stress for the adjacent tissues attaching thereto is moderate as shown by infrared-spectroscopy . RESULTS: A comparison with nonirradiated samples revealed that CO2 laser irradiation reduced color penetration at the section to a minimum . Also, irradiation of the root-canal wall resulted in satisfactory sealing of the surface . These findings were supported by the results of the SEM examinations . CONCLUSIONS: CO2 laser treatment optimally prepares the tooth for final intraoperative filling because of sealing of the dentinal tubules, the resultant elimination of niches for bacteria and the sterilizing effect of the laser.

Microbiology, 1998 May, 144 ( Pt 5), 1189 - 96
Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats; Frothingham R et al.; Genetic loci containing variable numbers of tandem repeats (VNTR loci) form the basis for human gene mapping and identification, forensic analysis and paternity testing . The variability of bacterial tandem repeats has not been systematically studied . Eleven tandem repeat loci in the M . tuberculosis genome were analysed . Five major polymorphic tandem repeat (MPTR) loci contained 15-bp repeats with substantial sequence variation in adjacent copies . Six exact tandem repeat (ETR) loci contained large DNA repeats with identical sequences in adjacent repeats . These 11 loci were amplified in 48 strains to determine the number of tandem repeats at each locus . The strains analysed included 25 wild-type strains of M . tuberculosis, M . bovis, M . africanum and M . microti and 23 substrains of the attenuated M . bovis BCG vaccine . One of the five MPTR loci and all six ETR loci had length polymorphisms corresponding to insertions or deletions of tandem repeats . Most ETR loci were located in intergenic regions where copy number may influence expression of downstream genes . Each ETR locus had multiple alleles in the panel . Combined analysis identified 22 distinct allele profiles in 25 wild-type strains of the M . tuberculosis complex and five allele profiles in 23 M . bovis BCG substrains . Allele profiles were reproducible and stable, as demonstrated by analyses of multiple isolates of particular reference strains obtained from different laboratories . VNTR typing may be generally useful for strain differentiation and evolutionary studies in bacteria.

Microbiology, 1998 May, 144 ( Pt 5), 1181 - 8
Analysis of lipids reveals differences between 'Mycobacterium habana' and Mycobacterium simiae; Mederos LM et al.; Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae . Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC . The strains of 'M . habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II', GPL-II and GPL-III; group C contained GPL-II', GPL-II and GPL-III; group D did not contain any of these compounds . Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant . Mycolic acids were also similar by TLC and HPLC, and consisted of alpha-, alpha'- and ketomycolates . Partial structural analysis by MS carried out in strains 'M . habana' TMC 5135 and M . simiae ATCC 25275T revealed that alpha- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and alpha'-mycolates from 58 to 67 carbon atoms . The alpha- and ketomycolates belonged to several structural series, and minor variations were found between the two strain examined . The data obtained justified the synonymy between 'M . habana' and M . simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis . Most strains of 'M . habana' can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains.

Comp Immunol Microbiol Infect Dis, 1998 Apr, 21(2), 135 - 54
Actinobacillus pleuropneumonia serotype 2--effects on the interferon-alpha production of porcine leukocytes in vivo and in vitro; Wattrang E et al.; Effects of a bacterial infection on the IFN-alpha production in vivo and in vitro were studied in eight specific pathogen free pigs experimentally infected with Actinobacillus pleuropneumoniae . Clinically, the experimental infection was manifested as a febrile stage which lasted approximately one week and by signs of respiratory disease . The Aujeszky's disease virus (ADV) induced IFN-alpha production, assessed in whole blood cultures, was increased for the infected pigs during the febrile stage . Potentiating effects on the IFN-alpha production could be transferred to cultures of purified peripheral blood mononuclear cells with sera collected from the infected pigs during this period of time . Although the experimental infection with A . pleuropneumoniae did not induce any detectable amounts of IFN-alpha in serum or nasal secretion, both a phenol-extract and a heat-inactivated preparation of the bacteria induced low levels of IFN-alpha in cultures of purified PBMC . The interferogenic structures of the bacteria were not identified but there were indications that the bacteria induced IFN-alpha production in the same cell type as ADV.

Wien Med Wochenschr, 1998, 148(4), 74 - 7
{Significance of prion protein in transmission of prions and in pathogenesis of spongiform encephalopathies}; Raeber AJ et al.; Prion disease or transmissible spongiform encephalopathies are caused by novel pathogens termed prions . Unlike classical infectious agents such as viruses or bacteria, prions lack an independent genome and consist largely if not entirely of an abnormal form of the host-encoded prion protein . How prions multiply is not known . A wealth of experimental evidence supports an essential role for the host-encoded prion protein in susceptibility and pathogenesis of prion diseases and in the propagation and spread of prions . In addition, B lymphocytes have been found to play a crucial role in the neuroinvasiveness of prions.

Nucleic Acids Res, 1998 Jun 15, 26(12), 2837 - 42
Evolutionary conservation of histone macroH2A subtypes and domains; Pehrson JR et al.; Histone macroH2A is an unusual core histone that contains a large non-histone region, and a region that resembles a full length H2A . We examined theconservation of this novel structural arrangement by cloning chicken macroH2A cDNAs and comparing them to their rat counterparts . The amino acid sequences of the two known macroH2A subtypes are >95% identical between these species despite evolutionary separation of approximately 300 million years . The H2A region of macroH2A is completely conserved, and thus is even more conserved than conventional H2A in these species . The origin of the non-histone domain was examined by comparing its sequence to proteins found in bacteria and RNA viruses . These comparisons indicate that this domain is derived from a gene that originated prior to the appearance of eukaryotes, and suggest that the non-histone region has retained the basic function of its ancestral gene.

J Dermatol, 1998 Apr, 25(4), 242 - 5
Pyodermia chronica glutealis complicated by acromegalic gigantism; Nishijima S et al.; We report a case of pyodermia chronica glutealis complicated by acromegalic gigantism associated with hyperprolactinemia . The serum prolactin, growth hormone, adrenocorticotropic hormone, and 11-deoxycortisol levels were elevated, but the estradiol and dehydroepiandrosterone-sulphate levels were within normal limits . However, the testosterone level was very low . Histopathologically, we found sinus tracts and scarring in a specimen from the buttocks . We could not immunohistochemically detect clear androgen, growth hormone, or prolactin receptors at any site . The patient was a man with a height of 197 cm and weight of 140 kg, he had clinical features of active acromegaly such as excessive sweating and increased thickness of soft tissue . He was also diagnosed with diabetes mellitus . Under such conditions, bacteria could easily grow and lesions might have been aggravated by the heavy pressure from his weight, a possible causes of his pyodermia chronica glutealis.

Biochemistry, 1998 May 19, 37(20), 7096 - 102
A hydroxyl group at residue 216 is essential for catalysis by human thymidylate synthase; Williams AW et al.; Structural analyses of bacterial thymidylate synthases (TSs) implicate a serine residue corresponding to Ser216 in human TS in hydrogen bond networks that are involved in binding of the nucleotide substrate, 2'-deoxyuridylate (dUMP), and that stabilize a beta-bulge in the protein . Utilizing site-directed mutagenesis, 12 mutant proteins were created with substitutions at residue 216 . DNA complementation studies utilizing a TS-negative bacterial strain revealed that only one mutant, Thr216 TS, supports the growth of the bacteria in the absence of thymidine . Kinetic characterization of the mutant proteins revealed that all TSs except Thr216 TS exhibited kcat/Kms for dUMP that are 10(3)-10(4) times lower, relative to that of wild-type TS . In addition, Thr216 TS was the only mutant to bind the mechanism-based inhibitor, 5-fluoro-2'-deoxyuridylate (FdUMP), into a ternary complex . Ligand binding studies revealed that Kds for dUMP binding to two defective mutants, Ala216 and Leu216 TSs, are 12-16-fold higher than that of wild-type TS . The data are consistent with the hypothesis that serine at this relative position is involved in dUMP binding; however, the data indicate that Ser216 has effects on catalysis, in addition to effects on dUMP binding . Catalysis is initiated by nucleophilic attack of the active site cysteine of TS on dUMP . The reaction rates of cysteine residues with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) were slower for Ala216 TS than for wild-type TS.

Microbiologia, 1997 Dec, 13(4), 445 - 52
Sorption of metals by Chlorobium spp; Garcia-Gil J et al.; The capacity of two species of green phototrophic sulfur bacteria, Chlorobium limicola and C . phaeobacteroides, to sorb several metal ions (Mn2+, Fe2+, Ni2+, Cu2+, Zn2+, Cd2+ and Pb2+) has been tested in laboratory batch cultures at increasing concentrations up to 2,000 mumol/l . Except for nickel--which was not sorbed to bacterial cells--the rest of metals tested were bound in a fast and passive process, which was mathematically described by means of Freundlich isotherms models . The sorption capacity of the two species studied were found to be dependent on the metal involved, whereas no differences were observed in the sorption intensity, suggesting that in all cases the sorption process proceeds in a similar way . Further, the comparison of the sorption intensity values as well as the metal recovery index (Ri), for both species, revealed that C . phaeobacteroides was more efficient that C . limicola to attach metal ions . The ecological significance of this ability in the water column of some stratified lakes, where coinciding maxima of ferrous iron and green photosynthetic sulfur bacteria are frequently found, is discussed.

Appl Environ Microbiol, 1998 Jun, 64(6), 2096 - 104
Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA (16S rDNA) and 16S-23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic fingerprinting, and partial 16S rDNA sequencing; Vinuesa P et al.; We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain . These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers . Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp . strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia . In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints . Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns . The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification . Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B . japonicum/Rhodopseudomonas rDNA cluster . Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C . proliferus, Macroptilium atropurpureum, and Vigna unguiculata.

Plant Cell, 1998 May, 10(5), 699 - 711
Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase; Tokuhisa JG et al.; Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC) . We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance . The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development . Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones . The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants . Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts . The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts . The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid.

Rheum Dis Clin North Am, 1998 May, 24(2), 261 - 73
Reactive arthritis; Schumacher HR Jr; Concepts about reactive arthritis are changing and must embrace consideration of the fact that bacteria or their products are present in the joint, not just at the portal of entry in the gastrointestinal (GI) or genitourinary (GU) tracts . With chlamydia-associated disease, atypical elementary bodies can be seen in synovium by electron microscopy, and nucleic acids, including RNA, can be found . It is not yet clear if bacterial nucleic acids are present in postenteric reactive arthritis and whether disease courses are predictably different after GI or GU infection . How bacteria are disseminated to joints and local factors, including cytokines that influence their persistence, are under study . Treatment with antibiotics may help some chlamydia-associated reactive arthritis but is not invariably effective.

Surg Today, 1998, 28(5), 529 - 37
Monitoring of acute allograft rejection by cytological, immunocytochemical, and immunohistochemical studies following rat small-bowel transplantation; Rahman MS et al.; We investigated the role of graft luminal fluid cytology for immunological monitoring of rat small-bowel allograft recipients . Allogeneic transplantation from WKAM (RT1u) to Lewis recipients and syngeneic transplantation using Lewis (RT11) rats were carried out . Twenty centimeters of the proximal jejunum was transplanted as a Thiry-Vella loop . The luminal fluid on days 0, 3, and 6 was examined cytologically using Papanicolaou, periodic acid-Schiff, and Giemsa staining, and immunocytochemically with monoclonal antibodies for macrophages (ED1 and ED2) . Full thickness biopsies of graft tissue were evaluated by both immunofluorescence (ED1 and ED2) and by standard histological methods . The cytological examination on day 6 revealed an increase in the number of enterocytes, lymphocytes, and neutrophils, the presence of bacteria, and the depletion of goblet cells in the allografts . Histologically, significant morphological changes of acute rejection were first seen on day 6 . Immunofluorescence predicted the acute rejection of the allografts earlier than a histological examination by showing an increase in the number of ED1- and Ed2-positive cells on day 3 . Graft luminal fluid cytology and immunofluorescence analysis of ED1 and ED2 cells could thus be used to recognize early acute allograft rejection following small-bowel transplantation.

FEBS Lett, 1998 May 8, 427(2), 291 - 5
A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium; Lee HM et al.; In the cyanobacterium Synechococcus sp . strain PCC 7942, ammonium exerts a rapid and reversible inhibition of the nitrate and nitrite uptake, and the PII protein (GlnB) is differentially phosphorylated depending on the intracellular N/C balance . RNA/DNA hybridizations, as well as nitrate and nitrite uptake experiments, were carried out with the wild-type strain and a PII-null mutant . The transcriptional control by ammonium of the expression of the nir-nrt ABCD-narB operon remained operative in the mutant but, in contrast to the wild-type strain, the mutant took up nitrate and nitrite even in the presence of ammonium . Moreover, the wild-type phenotype was restored by insertion of a copy of the wild-type glnB gene in the genome of the PII-null mutant . These results indicate that the unphosphorylated form of PII is involved in the short-term inhibition by ammonium of the nitrate and nitrite uptake in Synechococcus sp . strain PCC 7942.

Arch Biochem Biophys, 1998 May 15, 353(2), 322 - 30
An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum; Gutierrez-Cirlos EB et al.; Polytomella spp . is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall . A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutierrez-Cirlos et al., 1994, J . Biol . Chem . 269, 9147-9154) . Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp . exhibits an atypical alpha-band with a maximum at 567 nm . This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms . Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp . and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes . The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV . The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum . Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum . The data also show that the low potential bL heme from Polytomella spp . is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm . The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.

Cell Tissue Res, 1998 May, 292(2), 311 - 23
Distribution of murine mannose receptor expression from early embryogenesis through to adulthood; Takahashi K et al.; The mannose receptor is a 175-kDa transmembrane glycoprotein that appears to be expressed on the surface of terminally differentiated macrophages and Langerhans cells . The ectodomain of the mannose receptor has eight carbohydrate recognition domains . The receptor recognizes the patterns of sugars that adorn a wide array of bacteria, parasites, yeast, fungi, and mannosylated ligands . Clearance studies in whole animals have localized radiolabeled ligands, such as mannosylated bovine serum albumen, not only to macrophages, but also to liver sinusoidal endothelial cells . Hitherto, there has been no comprehensive analysis of expression of the mannose receptor in embryonic and adult mouse tissues . In this study, we have undertaken a systematic survey of the expression of the mannose receptor from early embryogenesis through to adulthood . The mannose receptor is expressed on tissue macrophages throughout the adult mouse as expected . However, the mannose receptor is first observed on embryonic day 9 on cells that line blood island vessel walls in the yolk sac . The mannose receptor is localized on sinusoidal endothelial cells in embryonic liver by embryonic day 11 and in bone marrow at embryonic day 17 . This pattern persists in these organs throughout embryogenesis into adulthood when sinusoidal endothelial cells of lymph nodes also express the mannose receptor . The receptor is also found on lymphatic endothelial cells of small intestine . In contrast, sinusoids of spleen and thymus do not express mannose receptor antigen . This study demonstrates that the mannose receptor is expressed on tissue macrophages and on subsets of vascular and lymphatic endothelial cells . Thus, the mannose receptor maybe a marker of the so-called reticuloendothelial system.

Arch Microbiol, 1998 May, 169(5), 424 - 33
A periplasmic flavoprotein in Wolinella succinogenes that resembles the fumarate reductase of Shewanella putrefaciens; Simon J et al.; During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens . However, the periplasmic cell fraction of W . succinogenes did not catalyze fumarate reduction with viologen radicals . W . succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria . The antigen was most likely encoded by the fccA gene of W . succinogenes . The antigen was absent from a DeltafccABC mutant, and its size is close to that of the protein predicted by fccA . The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide . The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S . putrefaciens fumarate reductase . As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC . The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c . FccB is similar to the N-terminal part (150 residues) of S . putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family . The DeltafccABC mutant of W . succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor.

Arch Microbiol, 1998 May, 169(5), 371 - 80
The oxygen-responsive NIFL-NIFA complex: a novel two-component regulatory system controlling nitrogenase synthesis in gamma-proteobacteria; Dixon R; The high energetic requirements for nitrogen fixation and the extreme oxygen sensitivity of the nitrogenase enzyme impose physiological constraints on diazotrophy that necessitate stringent control of nitrogen fixation (nif) gene expression at the transcriptional level . In the gamma-subdivision of the Proteobacteria, this control is maintained by a regulatory complex comprising an enhancer-binding protein (NIFA), which activates transcription at sigmaN-dependent nif (nitrogen fixation) promoters, and a sensor protein (NIFL), which inhibits NIFA activity in response to fixed nitrogen and external concentrations of molecular oxygen . Inhibition of NIFA activity by NIFL apparently requires stoichiometric amounts of the two proteins, implying direct protein-protein interaction rather than catalytic modulation of NIFA activity . NIFL contains FAD as a prosthetic group and is a novel type of flavoprotein in which the oxidation state of the bound flavin acts as a molecular switch to control transcriptional activation by NIFA . The FAD-binding domain of NIFL contains a motif common to a large family of redox sensory proteins . In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP, suggesting that formation of the inhibitory complex might be regulated by the ATP/ ADP ratio . Proposed mechanisms for the inhibition of NIFA activity by NIFL are beginning to emerge.

Cell Tissue Res, 1998 Apr, 292(1), 129 - 35
Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells; Becker EL et al.; The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria . The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced . We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types . The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal . Liver hepatocytes and Kupffer cells are positive . Smooth muscle and endothelial cells are also generally positive . In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively . These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.

Clin Diagn Lab Immunol, 1998 May, 5(3), 313 - 8
Analysis of the humoral immune response to Chlamydia outer membrane protein 2; Mygind P et al.; The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied . Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment . To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547) . By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of both C . pneumoniae and C . trachomatis infections (P < 0.0001) . The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.

Protein Sci, 1998 May, 7(5), 1180 - 5
The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry; Bruce JE et al.; Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI) . Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB) . A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis . Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis . However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry . To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry . Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.

J Immunol, 1998 Jun 1, 160(11), 5221 - 30
Early preferential stimulation of gamma delta T cells by TNF-alpha; Lahn M et al.; Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity . Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation . Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS . However, gamma delta T cells responded more strongly to the bacteria and to LPS . In vitro LPS-stimulated human T cells showed a similar differential response pattern . We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses . The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75 . Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels . The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state . As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.

J Thorac Cardiovasc Surg, 1998 May, 115(5), 1172 - 8
Down-regulation of surface monocyte lipopolysaccharide-receptor CD14 in patients on cardiopulmonary bypass undergoing aorta-coronary bypass operation; Fingerle-Rowson G et al.; OBJECTIVES: Major operative trauma like aorta-coronary bypass operation may lead to postoperative immunodisturbance, putting the patient at an increased risk for infection and sepsis . The monocyte/macrophage system and the endotoxin receptor CD14 are important in the early recognition and elimination of invading bacteria . The aim of this study was to analyze changes in membrane-associated CD14 and soluble CD14 during and after cardiac involving cardiopulmonary bypass . METHODS: We studied numbers of leukocytes, monocytes, and monocyte subpopulations, expression of monocyte membrane-associated CD14 and plasma levels of soluble CD14 in 10 patients (63 +/- 8 years of age), who underwent elective cardiopulmonary bypass . RESULTS: Cardiopulmonary bypass induced marked postoperative monocytosis, which was maximal 20 hours after the operation (485 +/- 242 cells/microl before, 1080 +/- 264 cells/microl 20 hours after surgery) . Expression of membrane-associated CD14 on classical CD14++ monocytes decreased significantly by 40%, reaching a nadir 20 hours after surgery (p < 0.05) . At the time of maximal membrane-associated CD14 suppression, the levels of soluble CD14 measured by enzyme-linked immunosorbent assay were clearly increased (3.2 +/- 1.0 microg/ml before versus 5.6 +/- 1.0 microg/ml 20 hours after, p < 0.001) . No significant change of the percentage of small (alpha) and large (beta) forms of soluble CD14 was found . CONCLUSIONS: Cardiopulmonary bypass leads to reduced membrane-associated CD14 expression on peripheral blood monocytes and increased levels of soluble CD14 through shedding or secretion of membrane-associated CD14 from the cell surface . These findings indicate that bypass is associated with significant monocyte activation.

Br Med Bull, 1998, 54(1), 139 - 50
Helicobacter pylori and gastric inflammation; Bodger K et al.; H . pylori infection leads to gastric inflammation, characterised histologically by surface epithelial degeneration and infiltration of the gastric mucosa by acute and chronic inflammatory cells . H . pylori adherence, the production of a vacuolating cytotoxin and bacterial enzymes all contribute to epithelial damage . Recruitment and activation of immune cells in the underlying mucosa involves H . pylori chemotaxins, epithelial-derived chemotactic peptides (chemokines) such as IL-8 and GRO-alpha, and pro-inflammatory cytokines liberated by mononuclear phagocytes (TNF alpha, IL-1 and IL-6) as part of non-specific immunity . Antigen-specific cellular immunity results in a predominant Th1 lymphocyte response with an increase in IFN-gamma secreting T-helper cells, whilst humoral responses lead to the production of anti-H . pylori antibodies and complement activation . The complex network of cytokines implicated in these inflammatory responses include counter-regulatory elements such as IL-10 which may serve to damp down inflammation . Molecular mimicry of host structures by H . pylori, with the generation of specific immunity directed against self-antigens may also contribute to host injury . Progress in molecular biology has revealed considerable genomic diversity amongst H . pylori strains, with cag+ bacteria being associated with increased chemokine and cytokine responses and more severe degrees of gastric inflammation . Strain hetereogeneity may contribute towards the wide spectrum of disease manifestations encountered in clinical practice.

Toxicon, 1998 Jan, 36(1), 41 - 51
Identification and characterization of novel sodium channel toxins from the sea anemone Anthopleura xanthogrammica; Kelso GJ et al.; Six new toxins from the sea anemone Anthopleura xanthogrammica were identified using a molecular biological approach . Five of these novel isoforms resemble the 47 residue type I long polypeptides native to Anthopleura elegantissima, Anthopleura fuscoviridis and Anemonia sulcata, while one appears to be chimera of the two previously identified 49 residue toxins native to A . xanthogrammica . Four of these toxins were expressed in bacteria, purified and characterized by ion flux assays in RT4-B and N1E-115 cell lines expressing the cardiac and neuronal Na channel isoforms, respectively . The novel 47 residue toxin isoforms form a new subclass within the A . xanthogrammica neurotoxin family, although they are related to previously described anemone toxins . One of the three 47 residue toxins characterized, PCR2-10, enhances veratridine-dependent sodium uptake, displaying a K0.5 of 329 nM and 1354 nM in RT4-B and N1E-115 cell lines, respectively . The novel 49 residue toxin, PCR3-7, interacts with the sodium channel with even higher affinity, enhancing sodium uptake with a K0.5 of 47 nM and 108 nM in RT4-B and N1E-115 cells, respectively.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 5884 - 90
Photoactive yellow protein: a structural prototype for the three-dimensional fold of the PAS domain superfamily; Pellequer JL et al.; PAS domains are found in diverse proteins throughout all three kingdoms of life, where they apparently function in sensing and signal transduction . Although a wealth of useful sequence and functional information has become recently available, these data have not been integrated into a three-dimensional (3D) framework . The very early evolutionary development and diverse functions of PAS domains have made sequence analysis and modeling of this protein superfamily challenging . Limited sequence similarities between the approximately 50-residue PAS repeats and one region of the bacterial blue-light photosensor photoactive yellow protein (PYP), for which ground-state and light-activated crystallographic structures have been determined to high resolution, originally were identified in sequence searches using consensus sequence probes from PAS-containing proteins . Here, we found that by changing a few residues particular to PYP function, the modified PYP sequence probe also could select PAS protein sequences . By mapping a typical approximately 150-residue PAS domain sequence onto the entire crystallographic structure of PYP, we show that the PAS sequence similarities and differences are consistent with a shared 3D fold (the PAS/PYP module) with obvious potential for a ligand-binding cavity . Thus, PYP appears to prototypically exhibit all the major structural and functional features characteristic of the PAS domain superfamily: the shared PAS/PYP modular domain fold of approximately 125-150 residues, a sensor function often linked to ligand or cofactor (chromophore) binding, and signal transduction capability governed by heterodimeric assembly (to the downstream partner of PYP) . This 3D PAS/PYP module provides a structural model to guide experimental testing of hypotheses regarding ligand-binding, dimerization, and signal transduction.

J Virol, 1998 Jun, 72(6), 5174 - 81
DNA immunization with minigenes: low frequency of memory cytotoxic T lymphocytes and inefficient antiviral protection are rectified by ubiquitination; Rodriguez F et al.; Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria . In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins . Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization . We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective . Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection . However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific . In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors . Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency . Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation . We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection . Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.

J Clin Periodontol, 1998 Apr, 25(4), 311 - 5
Short chain carboxylic acids decrease human gingival keratinocyte proliferation and increase apoptosis and necrosis; Sorkin BC et al.; Epithelia are key barriers to infections . In periodontal disease, the gingival sulcular epithelium becomes ulcerated . In this report, we test the hypothesis that short-chain carboxylic acids (SCCA) inhibit keratinocyte proliferation, increase necrosis and apoptosis, and may thus promote ulceration . SCCA produced by bacteria are present at millimolar concentrations in the periodontal pockets of subjects with periodontal disease . SCCA concentrations are higher in subjects with severe disease than in those with mild disease, and are not detectable in healthy subjects . Cell proliferation is critical for maintenance of epithelial barrier function . All SCCA tested, when neutralized, decreased epithelial cell proliferation (as measured by 3H-thymidine incorporation) in a dose-dependent manner . We found that epithelial cell viability decreased with increasing SCCA concentrations, accounting at least partly for the decreased 3H-thymidine incorporation . For all conditions we tested, SCCA-induced apoptosis preceded and exceeded necrosis . While the molecular mechanism(s) for these effects remain to be determined, the results indicate that SCCA derived from caries- or periodontal disease-associated bacteria could alter gingival barrier function.

Biochim Biophys Acta, 1998 Apr 14, 1364(1), 1 - 16
The generation of proton electrochemical potential gradient by cytochrome c oxidase; Rottenberg H; Cytochrome c oxidase, the terminal oxidase of mitochondria and some bacteria, catalyzes the four electron reduction of oxygen, and generates a proton electrochemical potential gradient (Delta microH) . The recently determined structures of the bacterial and the bovine enzymes, together with studies of site directed mutants of a bacterial cytochrome c oxidase and a closely related ubiquinol oxidase, have greatly advanced our understanding of the mechanism by which oxygen reduction is coupled to the generation of Delta microH . Two different mechanisms contribute to the generation of Delta microH: protons that are consumed by the reduction of oxygen, are taken exclusively from the mitochondrial matrix ('consumed' protons), while other protons are translocated by the enzyme across the membrane ('pumped' protons) . It is suggested that both proton consumption and proton pumping are driven by the electrostatic charging of the enzyme reaction center by the reducing electrons . Proton consumption is suggested to result from the electrostatically driven ejection of hydroxyls into the matrix that is catalyzed by a tyrosine residue in the reaction center . Proton pumping is suggested to result from the electrostatically driven translocation of a glutamate residue near the reaction center, and is assisted by secondary acceptors that release the translocated protons .

Curr Biol, 1998 Apr 9, 8(8), R288 - 90
Tubulin family: kinship of key proteins across phylogenetic domains; Egelman EH; Atomic structures obtained by electron microscopy for tubulin, and by X-ray crystallography for bacterial FtsZ, show that the two proteins are highly homologous . The complementarity between such high-resolution studies and low-resolution reconstructions of microtubule complexes is clear, but controversy still abounds.

Curr Biol, 1998 Apr 9, 8(8), R266 - 9
Genomics: re-evaluation of translation machinery evolution; Koonin EV et al.; Experiments based on genome sequence analysis have revealed unexpected complexity in the evolution of the translation apparatus, including concerted evolution of Gln-tRNA synthetase and Glu-tRNAGln amidotransferase, and a novel, class I Lys-tRNA synthetase shared by archaea and spirochaetes.

Foot Ankle Int, 1998 Mar, 19(3), 166 - 8
Results of preprocedure and postprocedure toe cultures in orthopaedic surgery; Zacharias J et al.; This study was to determine whether there is any benefit to wrapping the toes sterilely during orthopaedic procedures not involving the foot but performed on the lower extremity . The group studied consisted of 12 patients who had an orthopaedic procedure performed in which the foot and toes were included in the surgical prep, but not involved in the surgical procedure . Nine of the 12 patients (75%) had positive results from preprocedural aerobic cultures and two of the 12 (16.6%) had positive results from preprocedural fungal cultures . Recolonization of the bacteria between the toes was also demonstrated . Sterile draping of the toes would minimize the risk of infection and also protect against bacteria that recolonize during the procedure.

Rheumatol Int, 1998, 17(5), 193 - 6
The demographic and clinical spectrum of Arab versus Asian patients with ankylosing spondylitis in the UAE; al Attia HM et al.; Ankylosing spondylitis is a rather uncommon condition in the UAE . Over a period of 10 years . 28 hospital-based patients diagnosed as having AS were retrospectively studied . They included 17 Arabs and 11 Asians . The onset of AS in most patients in this study was in adulthood (mean age at onset was 27.7 years in Arabs and 28.75 years in Asians) . HLA B27 was positive in 56 and 81% in these two populations, respectively (P > 0.05) . Analysis of these figures, however, along with previous relevant published data, could indicate that Arabs with AS are less likely to be B27-positive than Asians . Among the Arab patients there was not a single case from the local community, which could be attributed to the extremely low rate of B27 phenotype in their normal population . The interracial variations in the frequency of clinical features were statistically insignificant, therefore indicating some degree of similarity in the form and disease expression in both groups . AS is characterized as being predominantly axial in the majority of our patients . Extraspinal (oligo-poly) arthropathy involved mainly hips and knees, and there have been fewer extra-articular manifestations compared with other series published.

J Gen Virol, 1998 May, 79 ( Pt 5), 1281 - 8
Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus; Schmitt C et al.; Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes . Mutagenesis studies have implicated two non-structural viral proteins in the transmission process . These two proteins {the 29 kDa ('29K') protein and the 23K protein} were expressed in bacteria and used to raise antibodies . In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N . clevelandii plants . Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated . A PEBV mutant was constructed lacking the complete 23K coding sequence . The mutant was able systemically to infect Nicotiana spp . but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots . These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV . The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission . Western immuno-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.

Crit Rev Oral Biol Med, 1998, 9(2), 179 - 200
Immune defense mechanisms of the dental pulp; Jontell M et al.; Defense reactions of the dentin/pulp complex involve a variety of biological systems, in which the immune system plays a pivotal role . The knowledge of the organization and function of pulpal immunocompetent cells has been sparse, but in recent years a significant body of information of immune mechanisms in general has provided a footing for substantial new knowledge of the immune mechanisms of the dental pulp . The identification of pulpal dendritic cells (DCs) has generated research activities which have led to a concept of how an antigenic challenge may evoke a pulpal inflammatory response . Although DCs are not able to identify foreign antigens specifically, they provide necessary signals to activate T-lymphocytes which in turn will orchestrate other immunocompetent cells to mount the local immune defense of the dental pulp . The purpose of this review is to accent the organization and function of pulpal DCs and other tissue and cellular components and to provide a basis for how they may interact to instigate pulpal defense mechanisms.

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