Cytometry, 1998 Oct 1, 33(2), 188 - 96 Performance of calibration standards for antigen quantitation with flow cytometry; Lenkei R et al.; In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM) . An unified window of analysis (UWA) was established on three different instruments (EPICS XL {Coulter Corporation, Miami, FL}, FACScan and FACS Calibur {Becton Dickinson, San Jose, CA}) with QC3 microbeads (FCSC, PR) . By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter {Immunotech}, and DAKO) was tested . In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared . mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument . Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses . Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition . Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators . The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols. Cytometry, 1998 Oct 1, 33(2), 179 - 87 Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence; Serke S et al.; Three types of microbead calibrators available for quantitative fluorescence flow cytometry have been studied in parallel using a variety of monoclonal antibodies (MoAbs) . The QIFI kit is designed for indirect immunofluorescence (IF), and both the Quantum Simply Cellular (QSC) assay and the Quanti-BRITE assay are designed for direct IF . Because of the different nature of the respective ligands, epitopes on cells versus F'ab-portions on QSC beads, large differences in titration curves for a large number of CD MoAbs were noted between QSC beads and cells . Use of the QSC assay and fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugates of the same CD reagent revealed substantially different numbers of cellular binding sites . Numbers of cellular binding sites as determined by direct IF using the Quanti-BRITE assay and by indirect IF using the QIFI kit were similar . We also found that erythrocyte (RBC)-lysing reagents cause varying and sometimes substantial reduction in the fluorescence intensity (FI) of cells stained directly with CD34 MoAb conjugates, but the RBC-lysing reagents had no effect on the FI of cells stained indirectly with the same CD34 MoAbs . This report defines a number of variables critical for standardized quantitative flow cytometry . We conclude that the choice of calibrators, fluorochrome conjugates, staining methods, and modes of sample processing can effect the determination of cellular binding sites to MoAbs . Direct immunofluorescence using the Quanti-BRITE assay and indirect IF using the QIFI kit appear to yield comparable results for the standardized determination of numbers of cellular binding sites to MoAbs. Cytometry, 1998 Oct 1, 33(2), 138 - 45 Quantitative flow cytometry: inter-laboratory variation; Zenger VE et al.; Quantitative flow cytometry (QFCM) offers a means of standardization within and between flow cytometers . QFCM parameters were set by determining the antibody-binding capacity (ABC) of CD4, CD8, and CD3 cells from 10 normal donors with the use of eight FACScan flow cytometers . QC3 beads and a certified blank bead were used to set up the instruments . Fluorescein isothiocyanate (FITC) conjugated to molecular equivalents of soluble fluorochrome (MESF) microbead standards was used before and after the donor samples were run to ensure that the machines were operating consistently . Lyophilized cells (Cytotrol) were used as a target, to control for antigen expression in the cell preparation . Quantitative Simply Cellular (QSC) beads were used to establish a standard calibration curve for each of the FITC and phycoerythrin antibody conjugates on each of the instruments . Single-parameter fluorescent histograms derived from list-mode files were used to calculate the slope (coefficient of response), intercept (zero channel), number of channels per decade, and ABC or MESF threshold (blank bead) . The fluorescence intensity (geometric mean) of the positive and negative donor cell populations was compared with the standard curves, and the ABCs were calculated . The results show consistent instrument performance between laboratories . However, after standardization of CD3, CD4, and CD8 ABCs to microbeads, large variations were noted between donors and laboratories . The source of this variation does not appear to be in the instrumentation but may be due to the lack of an unified set-up protocol, introducing issues of antibody saturation, methods for whole blood lysis and fixation, and the behavior of the microbead standards. Immunology, 1998 Aug, 94(4), 560 - 8 The regulation of superoxide generation and nitric oxide synthesis by C-reactive protein; Ratnam S et al.; Activated macrophages utilize both reactive oxygen intermediates and reactive oxynitrogen intermediates for defence against microbes . However, simultaneous generation of superoxide (O- 2;) and nitric oxide (NO) could be harmful to host cells due to the production of peroxynitrite, nitrogen dioxide and hydroxyl radicals . Therefore, the regulation of the production of these molecules is critical to host survival . During periods of inflammation or infection, the level of serum C-reactive protein (CRP) increases in many species . Human and rat CRP have been shown to bind and interact with phagocytic cells . Since many of the interactions of CRP involve the binding to the phosphocholine ligand, we studied the role of CRP in O- 2; and NO generation through the modulation of phosphatidylcholine (PC) metabolism in macrophages . This study has shown that, while rat CRP inhibited phorbol myristate acetate- (PMA) induced release of O- 2; by rat macrophages, CRP-treated macrophages released NO in a time- and dose-dependent manner . CRP increased inducible nitric oxide synthase (iNOS) enzyme as well as iNOS mRNA levels in rat macrophages . Tricyclodecan-9-yl-xanthogenate (D609), an inhibitor to PC phospholipase C (PC-PLC), suppressed iNOS induction but enhanced PMA-induced release of O- 2; . These data indicate that an increased level of CRP during periods of inflammation may result in differential regulation of macrophage NADPH oxidase and iNOS activity . Increased hepatic synthesis of CRP may contribute to the mechanism by which phagocytic cells avoid simultaneous O- 2; and NO synthesis, and this could possibly be mediated through the regulation of PC-PLC. J Mol Med, 1998 Sep, 76(10), 676 - 81 Clinical manifestation of myeloperoxidase deficiency; Lanza F; Myeloperoxidase (MPO), an iron-containing heme protein localized in the azurophilic granules of neutrophil granulocytes and in the lysosomes of monocytes, is involved in the killing of several micro-organisms and foreign cells, including bacteria, fungi, viruses, red cells, and malignant and nonmalignant nucleated cells . Despite the primary role of the oxygen-dependent MPO system in the destruction of certain phagocytosed microbes, subjects with total or partial MPO deficiency generally do not have an increased frequency of infections, probably because other MPO-independent mechanism(s) for microbicidal activity compensate for the lack of MPO . Infectious diseases, especially with species of Candida, have been observed predominantly in MPO-deficient patients who also have diabetes mellitus, but the frequency of such cases is very low, less than 5% of reported MPO-deficient subjects . Evidence from a number of investigators indicates that individuals with total MPO deficiency show a high incidence of malignant tumors . Since MPO-deficient PMNs exhibit in vitro a depressed lytic action against malignant human cells, it can be speculated that the neutrophil MPO system plays a central role in the tumor surveillance of the host . However, any definitive conclusion on the association between MPO deficiency and the occurrence of cancers needs to be confirmed in further clinical studies . Clinical manifestations of this disorder depend on the nature of the defect; an acquired abnormality associated with other hematological or nonhematological diseases has been occasionally described, but the primary deficiency is the form more commonly reported . Another area of interest pertinent to MPO expression is related to the use of anti-MPO monoclonal antibodies for the lineage assignment of acute leukemic cells, the definition of FAB MO acute myeloid leukemia, the identification of biphenotypic acute leukemias, and their distinction from acute leukemia with minimal phenotypic deviation . The advantage of MPO monoclonal antibodies over the MPO cytochemical assay relies in the ability of the former method to recognize the enzymatically inactive precursor forms of MPO. Am J Med Sci, 1998 Oct, 316(4), 264 - 70 Infectious agents as triggers of reactive arthritis; Wuorela M et al.; Reactive arthritis was originally defined as a sterile joint inflammation after infection elsewhere in the body, but this view has been challenged in the past decade since different antigens and DNA and RNA of various triggering microbes have been shown to exist at the sites of inflammation in the joints . It has been suggested that microbial antigens, or intact pathogens, are important for the pathogenesis of reactive arthritis, at least in the early phase of the disease, but the exact mechanism of how the pathogens contribute to the development of this usually self-limiting polyarthritis has not been discovered . This article reviews the theories on the role of infectious agents as triggers of reactive arthritis. Cell Mol Biol (Noisy-le-grand), 1998 Jul, 44(5), 735 - 46 Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine; Schutze K et al.; The UV-laser microbeam has been established as a valuable tool in a wide area of molecular biology as well as in medical research and applications . This system allows to cut or fuse microscopically small specimen . An important application of the cutting laser is laser microbeam microdissection (LMM) combined with laser pressure catapulting (LPC), which allows to procure single cells or small homogeneous cell areas for subsequent molecular analysis in an entirely "non-contact" manner . With LMM minute tissue areas, single cells or chromosomes are microdissected and separated from their surroundings . Subsequently, LPC ejects the dissectates directly into the cap of a sample tube without any mechanical contact . This enables the rapid procurement of homogeneous specimen from less than one up to several hundreds of micrometers in diameter without encroachment of the adjacent region . The mRNA information of the selected specimen as well as of the remaining probe are well preserved, as demonstrated with laser isolated samples from a routinely prepared tissue section of a differentiated colorectal adenocarcinoma . Reverse transcription of specific mRNA coding for cytoplasmic beta-actin and subsequent hemi-nested PCR amplification was not impaired . Any kind of tissue, as well as single cells from different sources and even subcellular structures can be captured using this laser method . Wherever homogeneous samples are required to analyze cell or chromosome-specific genetic alterations such as in cancer research or prenatal diagnosis this unique and rapid laser micropreparation method will become a key technology of great value. Cell Mol Biol (Noisy-le-grand), 1998 Jul, 44(5), 721 - 33 Laser tweezers are sources of two-photon excitation; Konig K; The most important application of continuous wave (cw) near infrared (NIR) microbeams in cellular and molecular biology are single-beam gradient force optical traps, also called "laser tweezers" . Laser tweezers have been used for optical picoNewton force determination as well as for 3D cellular and intracellular micromanipulation, such as optical spermatozoa transportation in laser-assisted in vitro fertilization . Light intensities in the MW/cm2 range are necessary to confine motile spermatozoa in the optical trap . The enormous photon concentration in space and time results in non-resonant two-photon excitation of endogenous and exogenous absorbers with electronic transitions in the ultraviolet and visible spectral range . Trap-induced two-photon excitation of intracellular flurorophores can be used to study metabolism and vitality of motile cells without additional fluorescence excitation sources . Therefore, laser tweezers as sources of two-photon excitation may act as novel non-linear tools in cell diagnostics . The far red/NIR trapping radiation, in particular <800 nm, may also excite endogenous absorbers such as NAD(P)H, flavins, porphyrins and cytochromes . Excitation of these cellular absorbers may result in oxidative stress via type I and type II photooxidation processes . Severe non-linear-induced cell damage in a variety of cells confined in <800 nm traps was found . Two-photon induced destructive effects are enhanced in multimode traps due to longitudinal mode-beating phenomena . Pulsed laser sources are not suitable for safe optical trapping of living cells . The use of single frequency long-wavelength NIR traps (800 nm-1200 nm) for vital cell handling is recommended. Cell Mol Biol (Noisy-le-grand), 1998 Jul, 44(5), 701 - 10 Laser tweezers and optical microsurgery in cellular and molecular biology . Working principles and selected applications; Greulich KO et al.; After focusing in a microscope, light can be used for micromanipulation of (sub-)micrometer sized objects . Focused beams of classical light ablate elements of the cell division machinery and switch the beating of hearts on a cellular basis . Focused lasers (laser microbeams or optical scissors) allow in addition very precise nanomachining in a wide field of applications, from developmental biology to plant biotechnology . While in microbeam work high power densities and efficient light-tissue interactions are required, optical tweezers work in a complementary way: Moderate power densities and small absorption of the laser by the biological material is needed . With light pressure and optical gradient forces optical tweezers can be used to move microscopic objects, even in the interior of closed cells . In total, most mechanical micromanipulation techniques known from cellular and molecular biology can be replaced by their optical correlate and some applications are possible which cannot be performed micromechanically . When these optical microtools are operated at their maximum performance, the physical effects are as interesting as their biological applications: The laser microbeam can generate extreme local temperatures, which however are dissipated within a few tens of nanoseconds and therefore cause damage only very locally . The optical tweezers with a working wavelength in the optical window of biological material (600-1100 nm) exert piconewton forces without any mechanical contact . The present article discusses some quantitative physical aspects of microbeams and optical tweezers and gives a few selected examples of applications. J Cell Biol, 1998 Oct 5, 143(1), 241 - 52 Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands; Wu X et al.; Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins . To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands . Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody . With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited . In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment . Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG . The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle . This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury. FASEB J, 1998 Oct, 12(13), 1255 - 65 Molecular mimicry and immune-mediated diseases; Oldstone MB; Molecular mimicry has been proposed as a pathogenetic mechanism for autoimmune disease, as well as a probe useful in uncovering its etiologic agents . The hypothesis is based in part on the abundant epidemiological, clinical, and experimental evidence of an association of infectious agents with autoimmune disease and observed cross-reactivity of immune reagents with host 'self' antigens and microbial determinants . For our purpose, molecular mimicry is defined as similar structures shared by molecules from dissimilar genes or by their protein products . Either the molecules' linear amino acid sequences or their conformational fits may be shared, even though their origins are as separate as, for example, a virus and a normal host self determinant . An immune response against the determinant shared by the host and virus can evoke a tissue-specific immune response that is presumably capable of eliciting cell and tissue destruction . The probable mechanism is generation of cytotoxic cross-reactive effector lymphocytes or antibodies that recognize specific determinants on target cells . The induction of cross-reactivity does not require a replicating agent, and immune-mediated injury can occur after the immunogen has been removed a hit-and-run event . Hence, the viral or microbial infection that initiates the autoimmune phenomenon may not be present by the time overt disease develops . By a complementary mechanism, the microbe can induce cellular injury and release self antigens, which generate immune responses that cross-react with additional but genetically distinct self antigens . In both scenarios, analysis of the T cells or antibodies specifically engaged in the autoimmune response and disease provides a fingerprint for uncovering the initiating infectious agent. Phys Med Biol, 1998 Sep, 43(9), 2491 - 501 Calculation of microplanar beam dose profiles in a tissue/lung/tissue phantom; Company FZ et al.; Recent advances in synchrotron generated x-ray beams with a high fluence rate permit investigation of the application of an array of closely spaced, parallel or converging microplanar beams in radiotherapy . The proposed technique takes advantage of the hypothesized repair mechanism of capillary cells between alternate microbeam zones, which regenerates the lethally irradiated endothelial cells . The lateral and depth doses of 100 keV microplanar beams are investigated for different beam dimensions and spacings in a tissue, lung and tissue/lung/tissue phantom . The EGS4 Monte Carlo code is used to calculate dose profiles at different depths and bundles of beams (up to 20 x 20 cm square cross section) . The maximum dose on the beam axis (peak) and the minimum interbeam dose (valley) are compared at different depths, bundles, heights, widths and beam spacings. Infect Immun, 1998 Oct, 66(10), 4651 - 5 Cytotoxic T-lymphocyte-mediated lysis of Toxoplasma gondii-infected target cells does not lead to death of intracellular parasites; Yamashita K et al.; CD8(+) T cells play a crucial role in the control of infection with intracellular microbes . The mechanisms underlying the CD8(+) T-cell-mediated clearance of the intracellular pathogen Toxoplasma gondii are, however, not completely understood . The effect of CD8(+) cytotoxic T-lymphocyte (CTL)-mediated lysis of host cells on the viability of intracellular T . gondii was investigated . Quantitative competitive PCR of the gene encoding T . gondii major surface antigen (SAG-1) was combined with treatment of the parasites with DNase, which removed the DNA template of nonviable parasites . The induction by CD8(+) CTLs of apoptosis in cells infected with T . gondii did not result in the reduction of live parasites, indicating that intracellular T . gondii remains alive after lysis of host cells by CTLs. J Theor Biol, 1998 Aug 21, 193(4), 691 - 708 Do microbes with peptides mimicking myelin cause multiple sclerosis if the T cell response to their unique peptides is limited? Kaufman MD. This hypothesis for the pathogenesis of multiple sclerosis is based upon assumptions about the response of the T cell repertoire to pathogens . Immunologic and epidemiologic observations of several conditions suggest that activation of T cells formed in early life mediate injury to the central nervous system . Early in life, selection of lymphocytes by the thymus produces a weakly autoreactive T cell repertoire which, with the help of transient maternally-derived defenses, recognizes pathogens . These responses later are supplemented by pathogen-specific responses, acquired as microbes are encountered . As the thymus involutes, the diversity of pathogen-specific responses to microbial epitopes is progressively fixed . Reduced and delayed pathogen exposure, common in developed societies, limits the repertoire of memory T cells, which can efficiently eliminate pathogens . Due to their small number, pathogen-specific lymphocytes which mature extrathymically may not be able to rapidly eliminate most pathogens, and without the editing of the thymus, they may be autoreactive . In this setting, novel pathogens with epitopes mimicking myelin may elicit a T cell response which is autoreactive . Peptides of common microbes are known to activate T cells recognizing dominant antigens of myelin . It is postulated that at the equator, intense, non-seasonal encounters with microbes elicit an immune repertoire that produces resistance to autoimmunity, while, in temperate climates, moderate, seasonal exposures increase susceptibility to it . The differences in responses to microbes between populations with a low or high prevalence of multiple sclerosis suggests that T cell repertoires are divergent in these groups . An exuberant innate response, postulated to diminish as the load of enteric microbes falls and sanitation improves in relation to the distance from the equator, may increase resistance to multiple sclerosis by eliminating the need for T cell activation . Human herpesvirus-6 and respiratory syncytial virus are possible prototypes of microbes which activate myelin-directed T cells. Nucleic Acids Res, 1998 Oct 1, 26(19), 4482 - 6 Concordance analysis of microbial genomes; Bruccoleri RE et al.; The set of proteins which are conserved across families of microbes contain important targets of new anti-microbial agents . We have developed a simple and efficient computational tool which determines concordances of putative gene products that show sets of proteins conserved across one set of user specified genomes and not present in another set of user specified genomes . The thresholds and the homology scoring criterion are selectable to allow the user to decide the stringency of the homologies . The system uses a relational database to store protein coding regions from different genomes, and to store the results of a complete comparison of all sequences against all sequences using the FASTA program . Using Web technology, the display of all the related proteins for a given sequence and calculation of multiple sequence alignments (using CLUSTALW) can be performed with the click of a button . The current database holds 97 365 sequences from 19 complete or partial genomes and 8798905 FASTA comparison results . A example concordance is presented which demonstrates that the target of the quinolone antibiotics could have been identified using this tool. Planta Med, 1998 Aug, 64(6), 551 - 4 Conjugates of Tremella polysaccharides with microbeads and their TNF-stimulating activity; Gao Q et al.; A mannan (A) and a heteroglycan (D) were prepared by partial acidic hydrolysis of T3, a major polysaccharide fraction of the fungus Tremella fuciformis Berk . Methylation analysis of A and D showed that they contained a 1-->3 linked mannosyl main chain, to which were linked different side chains at position 2, 4, or 6 of the mannosyl residues . A and D were conjugated to albumin microbeads (AM) by reductive amination . The conjugates showed significant cytokine-stimulating activity in vitro whereas unconjugated AM had no activity . Not conjugated A and D showed cytokine-stimulating activity only in about 100 times higher concentrations. Berl Munch Tierarztl Wochenschr, 1998 Jul-Aug, 111(7-8), 268 - 72 {Improvement and acceleration of the diagnosis of contagious bovine pleuropneumonia by direct detection of the microbe using polymerase chain reaction (PCR)}; Hotzel H et al.; A diagnostic procedure is described for the detection of Mycoplasma mycoides subsp . mycoides SC, the causative agent of contagious bovine pleuropneumonia . DNA extracted from clinical samples was investigated by the polymerase chain reaction (PCR) using at least 2 different primer pairs, one species-specific and another one specific for the class of Mollicutes . Using this method, the time required for detection of the pathogen was reduced to 2 days, whereas with traditional diagnostic methods (cultivation in broth, biochemical tests or immunofluorescence) the same finding would be available only within approximately 20 days . Although contagious bovine pleuropneumonia does not occur in Central Europe, there are occasional identifications of cattle having positive titres in the complement fixation test (CFT) . Immunoblotting analysis of such sera confirmed that the reason for this phenomenon were cross-reactions with taxonomically related mycoplasma species . The present PCR assay proved to be suitable because of its rapidity, as well as high specificity and sensitivity . In the case of positive serological findings it enables diagnosticians to provide evidence on the presence or absence of the agent at short notice. J Cell Sci, 1998 Oct, 111 ( Pt 20), 3073 - 80 Regulation of mRNA localization by transmembrane signalling: local interaction of HB-GAM (heparin-binding growth-associated molecule) with the cell surface localizes beta-actin mRNA; Fages C et al.; Localization of mRNAs is currently thought to be partially responsible for molecular sorting to specific compartments within the cell . In mammalian cells the best-studied example is the beta-actin mRNA that is localized to the cell processes, and its localization is necessary in migratory responses of cells . It is reasonable to assume that mRNA localization within cells is coupled to transmembrane signalling due to extracellular factors, but little is known about such putative mechanisms . We show here that HB-GAM, an extracellular matrix-associated factor that enhances migratory responses in cells, is able to localize beta-actin mRNA when locally applied to cells via microbeads . The HB-GAM-induced mRNA localization is specifically inhibited by low concentrations of heparin and by heparitinase treatment of cells, showing that cell-surface heparin-type glycans are required for the effect . The finding that soluble N-syndecan is also inhibitory suggests that the transmembrane proteoglycan N-syndecan, previously identified as an HB-GAM receptor, is involved in the mRNA-localizing effect of HB-GAM . Inhibition of the mRNA localization by the src-kinase inhibitor PP1 is compatible with an N-syndecan-mediated effect since the receptor function of N-syndecan has been recently found to depend on the src-kinase signalling pathway . The mRNA-localizing activity of N-syndecan is also suggested by the finding that affinity-purified anti-N-syndecan antibodies coated on microbeads are able to localize beta-actin mRNA. J Clin Microbiol, 1998 Oct, 36(10), 2810 - 6 Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate; Fredricks DN et al.; Molecular methods are increasingly used to identify microbes in clinical samples . A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors . Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason . The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media . Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol . Accordingly, SPS tends to copurify with DNA . An extraction method was designed for purification of DNA from blood culture media and removal of SPS . Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically . Successful amplification of the extracted E . coli 16S rRNA gene was achieved by adding 5 microliter of undiluted processed sample DNA to a 50-microliter PCR mixture . When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples . By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR . We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance. Can J Microbiol, 1998 Jun, 44(6), 521 - 7 Evidence that a deferrioxamine B degrading enzyme is a serine protease; Zaya N et al.; Siderophores are organic biomolecules synthesized by a wide variety of microbes . The molecules sequester ferric ion from environments where it is present at extremely low concentrations . Siderophores are of consequence with respect to microbial nutrition, pathogenicity, virulence, and microbe-plant interactions . How siderophores are degraded and returned to the carbon and nitrogen cycles is not well understood . The catalytic activity of an enzyme from a bacterium that degrades the siderophore deferrioxamine B has been examined . While the degradation of deferrioxamine B is sensitive to sulfhydryl and metal moiety inhibitors, the data presented is most consistent with the hypothesis that the enzyme uses a hydroxyl moiety (serine peptidase) to catalyze the degradation of deferrioxamine B . If sulfhydryl and metal inhibitors are simultaneously present at concentrations that when alone only partially inhibit the enzyme, the enzyme is unable to catalyze deferrioxamine B dissimilation . Analysis of the inhibitor experiments conducted led to the conclusion that the deferrioxamine B degrading enzyme is a serine-peptidase-like enzyme that needs calcium ions and sulfhydryl groups to be fully activated or stabilized . The knowledge of the catalytic moieties of the enzyme will be exploited to purify the enzyme. Arch Environ Contam Toxicol, 1998 Oct, 35(3), 417 - 25 Sensitivity to copper in a ciliate as a possible component of biological monitoring in the lagoon of venice Coppellotti O. The impact of copper was studied in cultures of the microbenthic organism Euplotes vannus (Ciliophora, Hypotrichida) . This ciliate was isolated from sediment collected from a particular area in the Lagoon of Venice, which may be considered almost unpolluted by heavy metals . The effects of copper exposure in the laboratory on growth, metal accumulation, total acid-soluble thiol levels, and glutathione levels were examined, together with morphological alterations . E . vannus exhibited tolerance toward copper up to a concentration exposure of 0.2 &mgr;g Cu/ml, at which cell growth rate overlapped that of controls . Copper accumulated up to 239 &mgr;g/g dry wt after exposure to 0.4 &mgr;g Cu/ml, and morphological alterations were evident in cells exposed to concentrations from this value upward . Processes of vegetative reorganization involving nuclear apparatus, membranelles, cirri, and cortex could be observed after 4 days of exposure to 0.4 &mgr;g Cu/ml . Laboratory experiments with cultures of ciliates of cosmopolitan distribution such as Euplotes species in controlled conditions indicated the value of these organisms, which constitute a simple model for a monitoring system suitable for prediction in multicellular organisms. Bone Marrow Transplant, 1998 Sep, 22(5), 477 - 84 A comparative study on the efficacy of CD8-positive cells in enhancing allogeneic bone marrow engraftment: cell sorting vs microbead selection; Sen-Majumdar A et al.; We have used a superparamagnetic microbead selection system to positively select a murine bone marrow CD8+ cell population . The functional ability of these cells to enhance allogeneic bone marrow engraftment was compared with that of fluorescence activated cell sorter purified CD8+ cells . The CD8+ cell population prepared by the microbead selection procedure was as effective as cell sorter purified CD8+ cells in enhancing T cell-depleted allogeneic bone marrow engraftment in lethally irradiated mice . Phenotypic characterization of these cells shows that most of these CD8+ cells express CD3 and the T cell antigen receptor complex. Ophthalmologica, 1998, 212 Suppl 1, 79 - 81 Conservative treatment of adnexal, epibulbar and intraocular neoplasms by means of electron microbeams: a preliminary study; Fantini M et al.; At present, episcleral plaque brachytherapy and charged particle or photon irradiation are the most commonly employed methods in ocular and adnexal conservative treatments . In the near future, a different therapeutic approach to these malignancies could be represented by a new device based on electron beam emission . The equipment used consists of an electron accelerator originally developed for intraoperative radiotherapy, modified to fit ocular pathologies . Adequate collimators and an enhanced mechanical accuracy are required for this special practice . An operative planning for epibulbar, adnexal and intraocular tumors is described, discussing its rationale for possible applications . Experimental tests using phantoms and Gafchromic(R) films are in progress . As all conservative treatments, the main gaol of this activity is the maintenance of a good visual function. Microbiol Mol Biol Rev, 1998 Sep, 62(3), 597 - 635 Molecular and biotechnological aspects of microbial proteases; Rao MB et al.; Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications . They perform both degradative and synthetic functions . Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms . Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation . Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively . They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site . Proteases play a critical role in many physiological and pathophysiological processes . Based on their classification, four different types of catalytic mechanisms are operative . Proteases find extensive applications in the food and dairy industries . Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies . There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents . Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application . Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme . Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships . Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes . Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology. Am J Clin Pathol, 1998 Sep, 110(3), 321 - 6 Automated cell count in flow cytometry: a valuable tool to assess CD4 absolute levels in peripheral blood; Bene MC et al.; The enumeration of lymphocyte subsets in absolute counts has long relied on different methods applied separately to whole blood cell count, lymphocyte differential appreciation, and flow cytometric evaluation of lymphocyte subsets percentages . The development of multicolor labeling methods inflow cytometry now allows a more homogeneous appreciation of several cell subsets among gated lymphocytes . The use of internal calibrators, such as microbead suspensions, also permits a direct appreciation of subsets in absolute counts in a single-platform method . These methods were compared with a traditional multiplatform method of assessing absolute counts of lymphocyte subsets in a pilot study in which all manipulations were performed by 1 person and in a full-scale larger study performed in the normal working conditions of a hospital laboratory . Microspheres seem to be a reliable tool to perform absolute count enumeration inflow cytometry, but several precautions in the sample preparation and flow cytometric analysis are required. Am J Physiol, 1998 Sep, 275(3 Pt 2), R677 - 82 Decompression sickness risk in rats by microbial removal of dissolved gas; Kayar SR et al.; We present a method for reducing the risk of decompression sickness (DCS) in rats exposed to high pressures of H2 . Suspensions of the human colonic microbe Methanobrevibacter smithii were introduced via a colonic cannula into the large intestines of the rats . While the rats breathed H2 in a hyperbaric chamber, the microbe metabolized some of the H2 diffusing into the intestine, converting H2 and CO2 to methane and water . Rate of release of methane from the rats, which was monitored by gas chromatography, varied with chamber H2 pressure . This rate was higher during decompression than during compression, suggesting that during decompression the microbe was metabolizing H2 stored in the rats' tissues . Rats treated with M . smithii had a 25% (5 of 20) incidence of DCS, which was significantly lower (P < 0.01) than the 56% (28 of 50) incidence of untreated controls, brought on by a standardized compression and decompression sequence . Thus using a microbe in the intestine to remove an estimated 5% of the body burden of H2 reduced DCS risk by more than one-half . This method of biochemical decompression may potentially facilitate human diving. Pediatr Pulmonol, 1998 Aug, 26(2), 129 - 34 Comparison of rapid bolus instillation with simplified slow administration of surfactant in lung lavaged rats; Fernandez-Ruanova MB et al.; The aim of this study was to compare the effects of modified porcine surfactant (Curosurf) given either by a simplified slow delivery technique or by the standard bolus method, on pulmonary gas exchange, lung mechanics, and surfactant distribution in rats with respiratory failure produced by lung lavage . Twelve rats with respiratory failure induced by lung lavage received 200 mg x kg(-1) body weight (b.w.) of tagged porcine surfactant, either by the standard bolus delivery technique or by a simplified 1-min intratracheal infusion method, not requiring interruption of mechanical ventilation . Cardiovascular parameters, arterial blood gases, and pulmonary mechanics were measured repeatedly . Surfactant distribution was also measured by dye-tagged microbead spheres . After surfactant administration, there were no overall major differences between groups in mean heart rate, blood pressure, arterial blood gases, dynamic lung compliance, respiratory system resistance, and pulmonary distribution of exogenous surfactant . However, after 180 min pulmonary gas exchange was better and compliance higher in the bolus than the 1-min infusion group . A transient decrease in blood pressure and heart rate was observed in the bolus group; this side effect was not seen in animals treated with the simplified 1-min infusion method . We conclude that in rats subjected to lung lavage, the infusion of porcine surfactant by a simplified 1-min procedure produced similar short-term effects compared to the same dose of surfactant given by the bolus method . We speculate that tracheal bolus dosing is highly effective and might be the preferable delivery method for porcine surfactant . Dosing by the simplified method described appears less effective, but since no significant differences were observed, and since it produced less acute adverse effects, it could be used when clinical circumstances preclude rapid delivery. Mol Cell Probes, 1998 Aug, 12(4), 243 - 7 Direct quantitation of HIV by flow cytometry using branched DNA signal amplification; Van Cleve M et al.; Adaptation of the branched DNA signal amplification technology to flow cytometry has resulted in a quantitative nuclei-acid assay with significant advantages over the microwell-based format . In this assay, microbeads, rather than microwell plates, are derivatized with nucleic-acid capture probes and the derivatized beads are used to capture single nucleic-acid targets, which then capture fluorescent reporter probes via branched DNA . The assay detects DNA or RNA targets, has a current lower sensitivity limit of 500 human immunodeficiency virus (HIV) RNA molecules and responds linearly to target level from 500 to at least 50,000 molecules . Since microbeads can easily interrogate large volumes, viral lysis and genomic RNA capture can proceed in one step from comparatively large volumes, and sample preparation is greatly simplified compared to the microwell-format bDNA assay. Immunol Res, 1998 Aug, 18(1), 41 - 53 Intestinal intraepithelial T lymphocytes . Our T cell horizons are expanding; Nanno M et al.; The alimentary tract is an essential structure for the ingesting of nutrients from the outside, and even most primitive animals have a straight tract that runs from the mouth to the anus . We come into contact with the outside world through our skin and mucous membranes . The surface area of the enteric mucous membrane, which absorbs nutrients, is enlarge through its ciliary structure, and the enteric cavity creates by far the largest external world that we come into contact with . For instance, the enteric mucosal surface of the human gastrointestinal tract covered by a single layer of epithelial cells corresponds to the size of one-and-a-half tennis courts, and the innumerable number of epithelial cells covering this mucous surface are entirely replaced by new epithelial cells in the space of just several days . Simultaneously, the fact that 60-70% of peripheral lymphocytes are congregating in the gastrointestinal tract supports the notion that the enteric mucous membrane represents an extremely dangerous locale, where numerous harmless/precarious external antigens come in through the wide array of food we injest on a daily basis, and the literally infinite amounts of normal intestinal flora intermingled from time to time with life-threatening microbes surge across . Surprisingly, approximately one out of the five cells in the intestinal epithelium are lymphocytes, most of which are ill-defined T cells having unusual, but distinctive characteristics and situated apparently so close to external antigens in the entire body . This article deals with the information that has been accumulated mainly in the past decade concerning the development, phenotypes, and possible function of these yet unacknowledged mucosal T cells that lurk in the anatomical front of the intestine. Surg Endosc, 1998 Sep, 12(9), 1137 - 40 Utility of insufflation filters in laparoscopic surgery; Frankel SM et al.; OBJECTIVES: To determine whether filters, regularly used as part of the insufflator tubing during laparoscopic surgery, trap microbial and particulate matter from CO2 tanks, thus preventing passage from one patient to another . METHODS: A total of 67 used filters were collected from 17 CO2 tanks and six insufflation machines at three local hospitals, and sterile unused filters were used as controls . The used filters were distributed equally and sequentially into three groups: Group I-viewed under a dissecting microscope for particulate matter; group II-examined by mass spectrometry for contamination with oils and other impurities; group III-incubated on sheep blood agar plates and evaluated for growth of microorganisms . RESULTS: Negative . Used filters were indistinguishable by all parameters from controls . CONCLUSIONS: This limited study suggests filters now used in laparoscopic surgery fail to trap microbes or particulate matter . The question remains whether tank waste is absent or these filters fail to trap waste matter. Aust N Z J Surg, 1998 Aug, 68(8), 592 - 8 Small vessel ischaemia induced by microbead embolization in the sheep hind limb; Margovsky A et al.; BACKGROUND: Peripheral ischaemia may be caused by small vessel disease but there has been no satisfactory experimental model for studying this condition . We have developed a model in which microbeads are embolized to the distal vascular bed of a sheep . This model induces ischaemia proportional to the volume of microbead infusion and allows the pathophysiology and therapy of small vessel occlusion to be studied . METHODS: Gradual reduction of femoral artery blood flow by 50% and 75% in unilateral hind limbs of eight sheep was achieved by slow introduction of latex microbeads (mean size = 400 microns) into the peripheral vascular bed . The other hind limb served as a control . Measurements of blood flow, subcutaneous temperature and arterial and venous blood gases were recorded in both hind limbs after each level of flow reduction . Angiography confirmed small vessel occlusion . Muscle samples were analysed for ultrastructural changes by transmission microscopy . RESULTS: A linear correlation was found between the amount of microbeads infused and the reduction in the blood flow . Significant subcutaneous temperature and venous pO2 changes were observed in the embolized limb at both 50% and 25% flow levels compared to baseline (P < 0.05, ANOVA) . Angiography demonstrated abrupt cut-off images of the small vessels . Transmission microscopy showed graded levels of muscle cell damage from ischaemia . CONCLUSIONS: Latex microbead embolization induces reproducible controlled small artery occlusion . The degree of outflow obstruction and the extent of ischaemia can be varied by delivering measured quantities of microbeads . This model should be useful for studying the pathophysiology of ischaemia and for assessing the efficacy of treatment, especially the use of pharmacological agents. Aust N Z J Surg, 1998 Aug, 68(8), 554 - 61 Ischaemia-reperfusion injury to the intestine; Kong SE et al.; Ischaemia-reperfusion injury (IRI) is of obvious relevance in situations where there is an interruption of blood supply to the gut, as in vascular surgery, or in the construction of free intestinal grafts . It is now appreciated that IRI also underlies the guy dysfunction that occurs in early shock, sepsis, and trauma . The events that occur during IRI are complex . However, recent advances in cellular biology have started to unravel these underlying processes . The aim of this review is to provide an outline of current knowledge on the mechanisms and consequences of IRI . Initially, IRI appears to be mediated by reactive oxygen metabolites and, at a later stage, by the priming and activation of polymorphonuclear neutrophils (PMN) . Ischaemia-reperfusion injury can diminish the barrier function of the gut, and can promote an increase in the leakage of molecules (intestinal permeability) or the passage of microbes across the wall of the bowel (bacterial translocation) . Ischaemia-reperfusion injury to the gut can result in the generation of molecules that may also harm distant tissues. J Parasitol, 1998 Aug, 84(4), 691 - 5 Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons; Ashton FT et al.; Parasitic nematodes of the genus Strongyloides are remarkable for their ability to switch between alternative free-living developmental pathways in response to changing internal environmental conditions . After exiting the host, soil-dwelling larval stages may develop either to infectivity via 2 microbiverous stages (homogonic development) or to free-living adulthood via 4 microbiverous larval stages (heterogonic development) . The progeny of these adults then give rise to the infective stage . In the latter case, free-living existence is extended in time and the number of infective larvae is greatly amplified . Anterior chemosensory neurons (amphidial neurons) are thought to respond to environmental cues and via signal transduction pathways control the direction of larval development . We now demonstrate by laser microbeam ablation that 2 classes of amphidial neurons (ASF and ASI), acting together, control the direction of free-living larval development . Larvae in which the neurons were killed developed to infectivity via the homogonic route rather than to adulthood via the otherwise predominant heterogonic route . These neurons are probable homologues of neurons ADF (=ASF) and ASI in Caenorhabditis elegans, suggesting the control of development at the cellular level is conserved among divergent taxa of nematodes . These observations also have important implications for the evolution of nematode parasitism and the design of new prophylactic measures against parasitic nematodes of medical and veterinary medical importance. J Immunol, 1998 Aug 15, 161(4), 1758 - 64 Granulysin-induced apoptosis . I . Involvement of at least two distinct pathways; Gamen S et al.; Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis . It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes . In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells . This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases . Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) . Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin . Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1 . These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways. Leuk Lymphoma, 1998 Aug, 30(5-6), 449 - 58 Fas antigen (CD95) expression in peripheral blood progenitor cells from patients with leukaemia and lymphoma; Anthony RS et al.; Fas antigen (CD95) is a cell surface receptor belonging to the tumour necrosis factor/nerve growth factor superfamily and is able to induce apoptosis when triggered by its' natural ligand or an anti-Fas antibody . Fas expression is low on CD34+ bone marrow (BM) progenitor cells, but is increased by various cytokines in vitro . We investigated Fas expression on CD34+ cells from 39 peripheral blood progenitor cell (PBPC) harvests and from 5 normal BM harvests by dual colour flow cytometry to determine if Fas expression was altered during mobilisation . By including calibrated microbeads during flow cytometry, we quantified the number of Fas antigen molecules per cell . A low percentage of PBPC (22%) and normal BM (23%) CD34+ cells expressed Fas antigen . Fas expression varied on CD34+ cells from different diseases and the highest expression was found in ALL (52%) . There was a significant three fold increase in the number of Fas molecules/cell expressed on CD34+ cells (PBPC 6,230 molecules/cell, BM 2,236; p = 0.0003) . This level of expression was considerably less than that for CD3/CD19 lymphocytes (33,095 molecules/cell) and CD14 monocytes (47,467 molecules/cell) in the PBPC harvest . In conclusion, mobilisation including the use of growth of factors, has minimal effect on CD34 progenitor cell Fas expression. J Dairy Sci, 1998 Jul, 81(7), 2029 - 39 Evaluation and application of the Cornell Net Carbohydrate and Protein System for dairy cows fed diets based on pasture; Kolver ES et al.; This study evaluated the Cornell Net Carbohydrate and Protein System for dairy cows consuming diets based on pasture, assessed the sensitivity of the model to critical inputs, and demonstrated application opportunities . Data were obtained from four grazing experiments and four indoor pasture feeding experiments (25 dietary treatments) involving dairy cows in New Zealand and the US . The model provided a reasonably good estimate of changes in body condition score (r2 = 0.78; slope not significantly different from 1), estimated energy balance (r2 = 0.76; slope not significantly different from 1), blood urea N (r2 = 0.94; underprediction bias of 0.5%), microbial N flow (r2 = 0.88; slope not significantly different from 1), and milk production . The model underpredicted dry matter intake (r2 = 0.80; 13% bias) and overpredicted ruminal pH (r2 = 0.47; 1.7% bias) . Predicted milk production was especially sensitive to changes in pasture lignin content, effective fiber, rate of fiber digestion, and amino acid composition of ruminal microbes . Milk production was first-limited by the supply of metabolizable energy when only high quality pasture was fed, but specific amino acids limited milk production when more than 20% of the diet consisted of a grain supplement . These results indicate that the Cornell Net Carbohydrate and Protein System can be used for dairy cows in a grazing system to make realistic predictions of performance. J Clin Pathol, 1998 May, 51(5), 364 - 9 Levels of expression of CD19 and CD20 in chronic B cell leukaemias; Ginaldi L et al.; AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias . METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry . Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies . Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC) . RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes . The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias . In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3) . In HCL there was strong CD19 expression (38 (16) x 10(3)) . In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias . CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders. Nat Biotechnol, 1998 Aug, 16(8), 737 - 42 Identification of expressed genes by laser-mediated manipulation of single cells; Schutze K et al.; We describe a rapid noncontact method for the capture of single cells or small tissue areas of any size or shape directly within the cap of a common microfuge tube . Prior to the laser-mediated transfer, the specimen is isolated by laser microbeam microdissection, forming a clear-cut gap around the selected area . Laser treatment does not impair subsequent RNA analysis . We have used this method to isolate a single cell from archival colon adenocarcinoma, and were able to detect point mutations within codon 12 of c-Ki-ras2 mRNA after nested RT-PCR analysis. Mol Microbiol, 1998 Jul, 29(1), 1 - 11 Meaning and therapeutic potential of microbial recognition of host glycoconjugates; Karlsson KA; The microbe-host interface is currently in focus because of attempts to develop infection therapy in humans based on either natural receptor saccharide (respiratory and gastrointestinal disease) or sophisticated sialic acid analogues designed from crystal structures (influenza) . Most of the known host receptors for microbes are glycoconjugates, and the diversity and selectivity of host tissue glycosylation allow for the tropism of infections . However, among the many binding specificities detected so far, the biological role has been proven only in a few infectious model systems . The existence of multiple specificities of a single microbe is both a complicating factor and a challenge, requiring expanded research with a special demand on glycoscience. J Cell Sci, 1998 Sep, 111 ( Pt 17), 2607 - 14 A cytoplasmic dynein required for mitotic aster formation in vivo; Inoue S et al.; An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca . Evidence is mounting that dynein is required for the formation of mitotic spindles and asters . Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole . Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters . To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N . haematococca, a dynein-deficient mutant was made . Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present . These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N . haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo. Braz J Med Biol Res, 1998 Jun, 31(6), 793 - 7 Precipitated immune complexes of IgM induce the generation of reactive oxygen species by rabbit polymorphonuclear leucocytes; Lucisano YM et al.; We report that immune complexes of IgM (ICIgM) antibodies and ovalbumin in the form of a precipitate from the equivalence zone induce the generation of reactive oxygen species by rabbit blood polymorphonuclear leucocytes (PMN), as measured by the chemiluminescence (CL) production in the presence of luminol . The kinetics of CL generation induced by ICIgM is quite different from that induced by precipitated immune complexes of IgG (ICIgG): the maximum rate of CL production for ICIgM occurs around 14 min, whereas for ICIgG it occurs about 5 min after incubation with the cells . Also the triggering of the process requires a higher concentration of ICIgM than of ICIgG . Evidence is presented that these effects are not mediated by interaction of the antigen (ovalbumin) with the cell, since immune precipitates of ovalbumin and the F(ab')2 fragment had no effect . Our observations that precipitated ICIgM can also be an effective stimulus for CL generation and thus for O2- production reveal a new functional capability of PMN . These results may have implications for the understanding of the participation of ICIgM (as well as of ICIgG) in inflammatory reactions mediated by PMN in immune complex diseases, and in the mechanisms of defense against microbes and other non-self agents. PDA J Pharm Sci Technol, 1998 May-Jun, 52(3), 89 - 99 Airborne contamination during blow-fill-seal pharmaceutical production; Whyte W et al.; The routes of airborne contamination, during Blow-Fill-Seal (BFS) production, were studied using tracer gas, particles and bacteria . The prevention of airborne contamination, by the air shower at the point of fill, was effective (> 99.2% efficient) . However, microbe-carrying particles could gain access, by deposition or air exchange, when the containers were cut open and before they shuttled under the protection of the air shower . The use of SF6 tracer gas demonstrated that when the air shower was not on, 50% of the air within the containers came from the area round the machine . When the air shower was switched on, only about 5% of the air came from the surroundings . Airborne microbial contamination of containers is in proportion to: the number of airborne microbes around the machine, the time the container is open, the neck area and the amount of air left within the container . The likely microbial contamination rate can be calculated from a model incorporating these variables . Microbial contamination of containers during BFS manufacturing is normally very low, but by increasing the naturally occurring bacteria in the air of the production rooms by about 100-fold, it was possible to verify the accuracy of this model . The contamination model agrees well with the observation that microbial contamination levels of between 1 in 10(5) and in 10(7) will be found when small containers (< 10 ml) are filled in conventionally ventilated rooms . To achieve similar contamination rates when filling of larger bottles, it is likely that unidirectional flow, or barrier technology will be required. Nutrition, 1998 Jul-Aug, 14(7-8), 573 - 9 Nutrition and the immune system of the gut; Cunningham-Rundles S et al.; Studies suggest that the development and expression of the regional immune system in the gastrointestinal (GI) tract is relatively independent of systemic immunity . This is reflected in significant differences in functional response of T cells and B cells and affects cytokine patterns and activation pathways when regional immunity is compared to systemic immunity . Nutrients have fundamental and regulatory influences on the immune response of the GI tract and, therefore, on host defense . In addition to the effect of nutrition during development, the local impact of different dietary and antigenic elements on the regional immune system contributes to potential diversion of the two systems throughout life . The route of exposure during antigenic contact is known to affect host immune response, whether it be a normal process, happening in the context of normal environmental encounter with nonpathogenic microbes or planned immunization, or occurring as a result of resolution of a potentially pathologic process i.e., an infectious encounter . Interactions at the local level profoundly influence systemic immune response, in part because of intrinsic differences in these systems, and also because of different requirements for optimal function . Although inflammatory processes are central to host defense in the periphery, the protective blocking action of the secretory immunoglobulin A immune response is crucial to local host defense, and, therefore, to the integrity of GI tract immune function . For these reasons, interaction with normal bacteria of the GI tract may be seen as the model of how the system has evolved and provide clues to the restoration of balance in the immunocompromised host . Reduction of normal commensal bacteria in the context of infection or after antibiotic treatment may interfere with nutrient availability and impair beneficial stimulation of GI immune response . This impairment may be associated with continued colonization with opportunistic microbes and inflammatory immune response that could lead to malabsorption and malnutrition . Study of the impact of nutrient imbalance on the function of the GI tract has profound implications for clinical medicine and may in the future lead to the rational design of preventive approaches to support immune response and host defense. J Pharm Biomed Anal, 1998 Aug, 17(4-5), 651 - 63 Quantitative imaging microscopy for the sensitive detection of administered metal containing drugs in single cells and tissue slices--a demonstration using platinum based chemotherapeutic agent; Mauthe RJ et al.; We described the use of Nuclear microscopy (microbeam PIXE) for the quantitative micron scale analysis of platinum based chemotherapeutic agents in individual cell and tissue slices . We demonstrate that microbeam PIXE has the sensitivity and accuracy to quantitatively measure the uptake of the chemotherapeutic agent cis-diamminedichloroplatinum (II) (cisplatin) and monitor other endogenous metal contents in single cells in a time- and dose-dependent fashion . Additionally, the technique can quantitatively image therapeutic levels of cisplatin and cisplatin analogs including cis-diammine{1,1-cyclobutanedicarboxylato} platinum (II) (carboplatin) in tissues from an animal model . This quantitative imaging microscopy has general application for the sensitive measurement of metal containing drugs/compounds at the cellular level and allows the study of cellular distribution and mechanism of action related to toxic response and cell function. Med Hypotheses, 1998 May, 50(5), 399 - 407 Can site-directed mutagenesis eliminate autoimmunity arising from molecular mimicry? Powell PD. One proposed mechanism of pathogenic retroviral infection involves autoimmunity . Molecular mimicry may occur between viral and host proteins which share sequence homologies . Immune processing of antigenic peptides can result in the generation of cross-reactive antibodies capable of binding to host tissues . Thus, it appears the immune system can inadvertently initiate an attack upon the host due to genetic similarities between non-self and self . Site-directed mutagenesis is a tool of molecular biology often utilized to induce genetic changes in a microbe of interest . Since retroviral etiology may possess an autoimmune component, it seems plausible to utilize site-directed mutagenesis to genetically shape the retroviral genome . Retroviruses possess a DNA intermediate in their lifecycle, allowing the problem of retroviral infection to be addressed as a genetic disorder of the host . Detrimental autoimmune responses associated with retroviral pathology might be ameliorated by shaping the genetic source of their existence. Emerg Infect Dis . 1998 Apr;4(2):351. Erratum Vol . 4, No . 1 Apoptotic repair of genotoxic tissue damage and the role of p53 gene. Atomic Energy Research Institute, Kinki University, Higashiosaka 577, Japan . skondo@taurus.bekkoame.or.jp The spontaneous mutation rate per replication per genome is nearly invariant in microbes; however, the rate of spontaneous genomic mutations in higher eukaryotes is much higher . Furthermore, the mutation rates per locus per generation among Drosophila, mice and humans are similar, despite the large differences in generation time . A simple explanation for these findings is that mice and humans have a specific antimutagenic mechanism that is lacking in Drosophila . I propose that apoptotic repair-deletion of genotoxic damage-bearing cells-operates in mammalian germ cells and that it works more accurately in humans than in mice because of a slower rate of cell turnover and a longer generation time . It has been a long-standing puzzle that germline mutation frequencies decrease markedly as the dose-rate of radiation is lowered in mice but not in Drosophila . This can be readily explained by p53-dependent apoptotic repair, because the p53 gene is absent from the genome of Drosophila . Fetuses of p53+/+ mice have proficient apoptotic repair capacity for X-ray-induced teratogenic damage, but p53-null fetuses completely lack this capacity . Further, I propose that the primary role of the p53 gene is to guard germ cells and embryos from genotoxic damage . This implies that the tumour suppressor function of the p53 gene results from p53-dependent apoptotic deletion of cells with genotoxic damage . The reasoning behind this proposal is given by reviewing reports that Drosophila larvae are insensitive to tumour formation after irradiation . Finally, I discuss the genetic effects of radiation in humans . Arch Tierernahr, 1998, 51(2-3), 225 - 35 Environmental effects on nutrient and energy metabolism in ruminants; Tamminga S et al.; Domesticated animals all over the world are subjected to a wide variety of environmental conditions and challenges . Any deviation from "normal" may result in adaptive behavior of which changes in feed intake or feed intake pattern is by far the most important . Adaptive behavior may further include influences on passage rate of feed residues through the digestive tract, resulting in changes in digestibility . Adaptive behavior may also result in changes in heat production, either to maintain body temperature constant, or as a result of an elevated body temperature . Important environmental challenges are infectious diseases . Mild (sub-clinical) infections usually result in reduced performance, without affecting feed intake or digestibility . Severe infections may disrupt the barriers between the internal metabolism and the respiratory and/or digestive tract, resulting in severe losses of energy and protein . This situation is notably apparent in severe infections with parasites of the gastrointestinal tract and may be associated with severe protein losses . Feeding high protein diets may partly alleviate the negative effects . Contamination of air, water and feed may occasionally cause problems in farm animals . Such contamination may include pathogenic microbes, toxic secondary fungal metabolites and heavy metals . Negative effects associated with such contamination often show an impaired reproductive efficiency, but their influence on the utilization of energy and nutrients is not well documented. J Immunol, 1998 Jul 15, 161(2), 868 - 76 Human dendritic cell (DC)-based anti-infective therapy: engineering DCs to secrete functional IFN-gamma and IL-12; Ahuja SS et al.; An imbalance in the Th1- and Th2-type cytokine responses may allow certain microbes to modify the host response to favor their own persistence . We now show that infection/pulsing of human CD34+ peripheral blood hemopoietic progenitor cell-derived dendritic cells (DCs) with Leishmania donovani promastigotes, Histoplasma capsulatum, and Mycobacterium kansasii impairs the constitutive production of IL-12 from these cells . Thus, strategies aimed at modulating a dysregulated Th1/Th2 response to infection would be of great interest . To both augment the host immune response and deliver potent immunomodulatory cytokines such as IL-12 and IFN-gamma, our goal is to develop a therapeutic strategy using genetically modified, microbial Ag-pulsed DCs . Toward developing such immunotherapies, we used retrovirus-mediated somatic gene transfer techniques to engineer human DCs to secrete biologically active IL-12 and IFN-gamma . DCs pulsed with microbial antigens (e.g., leishmania and histoplasma Ags) were capable of inducing proliferative responses in autologous CD4+ lymphocytes . CD4+ lymphocytes cocultured with IL-12-transduced autologous DCs had enhanced Ag-specific proliferative responses compared with CD4+ lymphocytes cocultured with nontransduced or IFN-gamma- transduced DCs . In this cell culture model system we demonstrate that IL-12 has a negative effect on IL-4 secretion that is independent of its ability to induce IFN-gamma secretion . Taken together, these results indicate that IL-12-transduced DCs may be specifically suited in inducing or down-modulating Ag-specific Th1 or Th2 responses, respectively, and thus may be useful as adjunctive therapy in those intracellular infections in which a dominant Th1 response is critical for the resolution of infection. J Acoust Soc Am, 1998 Jul, 104(1), 518 - 29 The perception of speech gestures; Surprenant AM et al.; Two experiments examined the effects of temporal overlap of speech gestures on the perception of stop consonant clusters . Sequences of stop consonant gestures that exhibit temporal overlap extreme enough to potentially eliminate the acoustic evidence of (at least) one of the consonants were obtained from x-ray microbeam data . Subjects were given a consonant monitoring task using stimuli containing stop sequences as well as those containing single stops . Results showed that (1) the initial consonant in the stop sequences was detected significantly less often than in the single stops; (2) bilabial gestures were considerably more effective at obscuring a preceding alveolar than the reverse; and (3) the detection rate correlated with an index of overlap between lip and tongue tip gestures . Experiment 2 employed stimuli that were truncated during the closure for the critical stop or stop sequence, so as to eliminate any information occurring in the acoustic signal at the stop release . This experiment showed that removing release information decreased detectability of the consonants generally . However, consistent with the observed gestural patterns, removing the release did not decrease detection of the alveolar stop when it was the first consonant of a sequence, indicating that there was no information about the alveolar stop present in acoustic realization of the second stop release . These experiments show that certain gestural patterns actually produced by English speakers may not be completely recoverable by listeners, and further, that it is possible to relate recoverability to particular metric properties of the gestural pattern. Ann N Y Acad Sci, 1998 Jun 30, 851, 3 - 12 The stress concept and neuroimmunoregulation in modern biology; Berczi I; Sixty years ago Hans Selye discovered that the neuroendocrine and immune systems interact during stress . The pathophysiological significance of neuroendocrine-immune interaction during injury has only been recognized recently . Today it is rapidly emerging that, in addition to defense against exogenous pathogenic agents, the immune system plays a key role in host defense against injury . During acute-phase reactions to infection/injury, when there is no time to mount a specific immune response, the neuroimmunoregulatory network suppresses specific immunity while rapidly elevating the production of acute-phase proteins (APP) in the liver . APP recognize microbes and abnormal cells/tissues and activates the immune system nonspecifically to fight infection or injury . There is a remarkable similarity between the stress syndrome as outlined by Selye in 1946 and the acute-phase response as we know it today . Moreover, it is becoming clear that the immune system participates in the normal physiological regulation of the body, which was also recognized by Selye in his later years . Although with much delay, the scientific community is beginning to fully appreciate Selye's ingenious discoveries which were far ahead of his time. Cytometry, 1998 Jul 1, 32(3), 214 - 22 Flow cytometric quantification of cyclin E in human cell lines and hematopoietic malignancies; Erlanson M et al.; Cyclins are important in the regulatory system controlling cell cycle progression and overexpression or aberrant expression of G1 cyclins, as cyclin E, may result in an inadequate G1-S transition . In this study, the expression of cyclin E was analyzed in 12 human cell lines and 18 hematological malignancies by flow cytometry, and protein levels were quantified using calibrating microbeads . The majority of the tested cell lines demonstrated a cell cycle-specific expression of cyclin E, even though some cell lines expressed lower and more constant cyclin E levels throughout the cell cycle . No correlation between the fraction of cyclin E-positive cells and cyclin E content could be observed in the cell lines . Similar cyclin E levels were observed in the tumor samples as for the cell lines, although the fractions of positive cells were low . The flow cytometric quantification of cyclin E was compared with densitometric quantification of cyclin E Western blots, and a significant correlation between the two methods was observed (rs = 0.56, P = 0.03) . The variation of cyclin E levels between cell lines was significantly higher than the variation for repetitive analyses of the same cell line, suggesting that the expression of cyclin E could reliably be quantified using flow cytometry . Because of the potential to analyze specific subgroups such as cyclin E-positive cells, this method may facilitate the detection of cyclin E-overexpressing tumor cells. Plant Physiol, 1998 Jul, 117(3), 1031 - 6 Systemin potentiates the oxidative burst in cultured tomato cells Stennis MJ, Chandra S, Ryan CA, Low PS. Plants that have been wounded by insects or other herbivores may be more susceptible to infection by adventitious microbes . Wound-induced signal molecules, which serve to induce responses in the plant that retard further feeding, might also act to prepare a plant for possible pathogen attack . We have examined the effect of a wound-generated systemic messenger (systemin) on a pathogen-stimulated defense-response marker, the oxidative burst . We observed that neither systemin nor its inactive analog (A-17) was able to directly induce H2O2 biosynthesis in suspension-cultured tomato (Lycopersicon esculentum L.) cells, regardless of the duration of exposure of the cells to the two peptides . Similarly, neither systemin nor A-17 was capable of modifying an oligogalacturonide-elicited oxidative burst, as long as elicitor addition occurred within minutes of treatment with systemin or A-17 . In contrast, preexposure of the cell cultures to systemin (but not to A-17) led to a time-dependent enhancement of the oligogalacturonide-elicited oxidative burst . By 12 h of exposure, the H2O2 biosynthetic capacity of systemin-treated cells exceeded that of the control cells by a factor of 16 +/- 2 . A similar up-regulation by systemin of a mechanically stimulated oxidative burst was also observed . Because the systemin-induced augmentation in oxidant synthesis is quantitatively prevented by coincubation with 2 &mgr;M cycloheximide, and because the oxidative burst of oligogalacturonic acid-elicited control cells (no systemin exposure) is unaffected by preincubation with cycloheximide, we conclude that systemin enhancement of the tomato-cell oxidative burst requires protein synthesis. J Acquir Immune Defic Syndr Hum Retrovirol, 1998, 18 Suppl 1, S20 - 4 Infection control basis for recommending one-time use of sterile syringes and aseptic procedures for injection drug users; Gershon RR; Persons who inject drugs are at increased risk for many infectious diseases, including HIV . Reuse of syringes and needles and other equipment used to prepare and inject drugs increases the risk of infection with blood-borne and other pathogens . According to standard infection control guidelines, needles and syringes and their contents fall within the critical category of patient contact because they enter the normally sterile vascular space . Medical equipment considered critical should be treated or prepared in a manner that destroys vegetative and spore-forming microbes . The simplest way to ensure this level of hygienic practice is to use prepackaged, sterile, disposable needles and syringes and to use sterile drug preparation equipment and drugs . These recommendations are made as part of a comprehensive infection prevention program designed for injection drug users. Semin Immunol, 1998 Jun, 10(3), 169 - 78 Microbial corruption of the chemokine system: an expanding paradigm; Pease JE et al.; The chemokine signaling system includes more than 40 secreted pro-inflammatory peptides and 12 G protein-coupled receptors that together orchestrate specific leukocyte trafficking in the mammalian immune system, ideally for anti- microbial defense and tissue repair processes . Paradoxically and perversely, some chemokines and chemokine receptors are also promicrobial factors and facilitate infectious disease, the result of either exploitation or subversion by specific microbes . Two modes of exploitation are known: usage of cellular chemokine receptors for cell entry by intracellular pathogens, including HIV, and usage of virally-encoded chemokine receptors for host cell proliferation . Likewise, two modes of subversion are known: virally-encoded chemokine antagonists and virally-encoded chemokine scavengers . Understanding how microbes turn the tables on the chemokine system may point to new methods to prevent or treat infection, or, more generally, to treat inappropriate chemokine-mediated inflammation . Lancet, 1997 Oct 25, 350(9086), 1240 - 4 Applications of molecular microbiology to vaccinology; Moxon ER; Genetics, cell biology, and whole-genome sequencing of pathogens have changed dramatically the opportunities to investigate the epidemiology, pathogenesis, diagnosis, and control of microbial diseases . For example, recombinant DNA and PCR are powerful tools used to isolate genes whose role in pathogenicity can be investigated in biologically relevant virulence assays . Vaccines that target one or more of these genes can then be developed . Complete genome sequences of microbes provide an inventory of the genes encoding every virulence factor and potential immunogen . Candidate vaccines can be selected and developed using various approaches, including the recent innovation of immunisation with nucleic acids . Although many successful vaccines have been and will continue to be developed through empirical approaches, molecular microbiology provides a rational basis for discovery, development, and implementation of safer, more effective and, potentially cheaper vaccines. Dev Comp Immunol, 1998 Mar-Apr, 22(2), 173 - 84 Intracellular signaling events in superoxide generation and adhesion of channel catfish, Ictalurus punctatus rafinesque, neutrophils to the extracellular matrix protein fibrinogen; Ainsworth AJ et al.; Activation of channel catfish neutrophils is essential if these cells are to participate in adhesion to extracellular matrix proteins or generate intracellular superoxide for killing of microbes . Various signaling pathways are required for these activities to occur . The objective of this study was to identify components of the signal transduction pathways in channel catfish neutrophils . A23187, bryostatin, and phorbol dibutyrate (PDBU) all induced catfish neutrophil adhesion to fibrinogen coated plates and the adhesion could be significantly reduced when neutrophils were pretreated with staurosporine (1 x 10(-7) M) . Staurosporine was the only inhibitor used in the study that inhibited or reduced PDBU-induced adhesion of catfish neutrophils to fibrinogen . Phorbol dibutyrate at the concentrations used in the adhesion assay was the only stimulant that caused generation of intracellular superoxide and therefore was the only stimulant used in the remainder of the study . Aristolochic acid (1 x 10(-4) and 3 x 10(-5) M) + PDBU and staurosporine (1 x 10(-7) and 1 x 10(-8) M) + PDBU caused a significant decrease (p < or = 0.05) in PDBU-induced intracellular oxygen generation . The role of protein kinase C and phospholipases in channel catfish neutrophil adhesion and superoxide generation are discussed. Rev Sci Tech, 1998 Apr, 17(1), 95 - 107 Porcine major histocompatibility complex; Vaiman M et al.; The major histocompatibility complex in swine (swine leucocyte antigen: SLA) is located on chromosome 7 with the class I and class III regions separated by the centromere from the class II region . The overall molecular organisation of the class I and III regions is well known, but further research is needed to establish that of the class II region . Approximately sixty genes have been characterised to date, including ten tightly packed SLA class I sequences . The exact number of functional polymorphic class I genes, as defined by serology, probably varies from one to four, depending on the haplotype . At least two other distantly class I-related gene families exist . The numerous and significant associations reported between SLA haplotypes and physiological traits are described . These traits include immune responsiveness to a variety of microbes and metazoan parasites, and male and female production and reproduction performance . The results obtained suggest that selection for specific SLA haplotypes may assist in the improvement of porcine production. Infect Immun, 1998 Jul, 66(7), 3029 - 34 Expression of Legionella pneumophila virulence traits in response to growth conditions; Byrne B et al.; In nature, Legionella pneumophila replicates exclusively as an intracellular parasite of amoebae, but it also persists in the environment as a free-living microbe . Studies of how this opportunistic pathogen recognizes and responds to distinct extracellular and intracellular environments identified a link between the growth phase and expression of traits previously correlated with virulence . When cultured in broth, only post-exponential-phase L . pneumophila was sodium sensitive, cytotoxic, osmotically resistant, competent to evade macrophage lysosomes, infectious, and motile . Likewise, the L . pneumophila phenotype changed during growth in macrophages . During the intracellular replication period, this bacterium was sodium resistant and lacked flagella; concomitant with macrophage lysis, L . pneumophila became sodium sensitive and flagellated . Expression of the virulent phenotype was a response to starvation, since exponential-phase L . pneumophila became cytotoxic, sodium sensitive, and motile after incubation in broth from stationary-phase cultures, except when it was supplemented with amino acids . Together, these data indicate that while nutrients are plentiful, intracellular L . pneumophila organisms are dedicated to replication; when amino acids become limiting, the progeny express virulence factors to escape the spent host, to disperse and survive in the aquatic environment, and to reestablish a protected intracellular niche favorable for growth. J Cell Biol, 1998 May 18, 141(4), 979 - 92 A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT); Pazour GJ et al.; Several enzymes, including cytoplasmic and flagellar outer arm dynein, share an Mr 8,000 light chain termed LC8 . The function of this chain is unknown, but it is highly conserved between a wide variety of organisms . We have identified deletion alleles of the gene (fla14) encoding this protein in Chlamydomonas reinhardtii . These mutants have short, immotile flagella with deficiencies in radial spokes, in the inner and outer arms, and in the beak-like projections in the B tubule of the outer doublet microtubules . Most dramatically, the space between the doublet microtubules and the flagellar membrane contains an unusually high number of rafts, the particles translocated by intraflagellar transport (IFT) (Kozminski, K.G., P.L . Beech, and J.L . Rosenbaum . 1995 . J . Cell Biol . 131:1517-1527) . IFT is a rapid bidirectional movement of rafts under the flagellar membrane along axonemal microtubules . Anterograde IFT is dependent on a kinesin whereas the motor for retrograde IFT is unknown . Anterograde IFT is normal in the LC8 mutants but retrograde IFT is absent; this undoubtedly accounts for the accumulation of rafts in the flagellum . This is the first mutation shown to specifically affect retrograde IFT; the fact that LC8 loss affects retrograde IFT strongly suggests that cytoplasmic dynein is the motor that drives this process . Concomitant with the accumulation of rafts, LC8 mutants accumulate proteins that are components of the 15-16S IFT complexes (Cole, D.G., D.R . Deiner, A.L . Himelblau, P.L . Beech, J.C . Fuster, and J.L . Rosenbaum . 1998 . J . Cell Biol . 141:993-1008), confirming that these complexes are subunits of the rafts . Polystyrene microbeads are still translocated on the surface of the flagella of LC8 mutants, indicating that the motor for flagellar surface motility is different than the motor for retrograde IFT. Biotechnol Prog, 1998 May-Jun, 14(3), 517 - 26 Characterization of the degradation of polylactic acid polymer in a solid substrate environment; Agarwal M et al.; Polylactic acid (PLA) polymer film was degraded in abiotic and biotic environments to understand the role of microbes in the degradation process of lactic acid based polymers . The degradation studies were conducted in a well-characterized biotic system, an abiotic system, a sterile aqueous system, and a desiccated environment maintained at 40, 50, and 60 degrees C . The combination of experiments in different environments isolated the distinct effect of microbes, water, and temperature on the morphological changes in the polymer during degradation . Due to lack of availability of radiolabeled PLA, various analytical techniques were applied to observe changes in the rate and/or mechanism of degradation . CO2 evolved, weight loss, and molecular weights were measured to evaluate the extent of degradation . X-ray diffraction and differential scanning calorimetry techniques monitored the morphological changes in the polymer . FTIR was used as a semiquantitative tool to gather information about the chemistry of the degradative process . Neither of the above analytical techniques indicated any difference in the rate or mechanism of degradation attributable to the presence of microorganisms . The extent of degradation increased at higher process temperatures . FTIR data were evaluated for significant statistical difference by t-test hypothesis . The results confirmed hydrolysis of ester linkage as the primary mechanism of degradation of PLA . On the basis of these data, a probable path of PLA degradation has been suggested. Biotechnol Prog, 1998 May-Jun, 14(3), 356 - 63 Micropatterned surfaces for control of cell shape, position, and function; Chen CS et al.; The control of cell position and function is a fundamental focus in the development of applications ranging from cellular biosensors to tissue engineering . Using microcontact printing of self-assembled monolayers (SAMs) of alkanethiolates on gold, we manufactured substrates that contained micrometer-scale islands of extracellular matrix (ECM) separated by nonadhesive regions such that the pattern of islands determined the distribution and position of bovine and human endothelial cells . In addition, the size and geometry of the islands were shown to control cell shape . Traditional approaches to modulate cell shape, either by attaching suspended cells to microbeads of different sizes or by plating cells on substrates coated with different densities of ECM, suggested that cell shape may play an important role in control of apoptosis as well as growth . Data are presented which show how micropatterned substrates were used to definitively test this hypothesis . Progressively restricting bovine and human endothelial cell extension by culturing cells on smaller and smaller micropatterned adhesive islands regulated a transition from growth to apoptosis on a single continuum of cell spreading, thus confirming the central role of cell shape in cell function . The micropatterning technology is therefore essential not only for construction of biosurface devices but also for the investigation of the fundamental biology of cell-ECM interactions. Pharm Res, 1998 May, 15(5), 661 - 70 Genetic vaccines: strategies for optimization; Gregoriadis G; Vaccination with attenuated or killed microbes, purified or recombinant subunit proteins and synthetic peptides is often hampered by toxicity, the presence of infectious agents, weak immune responses and prohibiting costs, especially in the developing world . Such problems may be circumvented by genetic immunization which has recently emerged as an attractive alternative to conventional vaccines . Numerous studies have already shown that immunization of experimental animals with plasmid DNA encoding antigens from a wide spectrum of bacteria, viruses, protozoa and cancers leads to protective humoral and cell-mediated immunity . This review deals with the background and progress made so far with DNA vaccines and their theoretical and practical advantages as well as potential risks, discusses proposed mechanisms of DNA transfection of cells and induction of immune responses to the produced vaccine antigen, and evaluates strategies for the control and optimization of such responses. Hum Immunol, 1998 May, 59(5), 313 - 22 Simultaneous HLA Class I and Class II antibodies screening with flow cytometry; Pei R et al.; A flow cytometric method of simultaneously screening both HLA Class I and Class II panel reactive antibodies (PRA) was developed using a pool of 30 different Class I and 30 different Class II microbeads coated with purified HLA antigens . The antibodies in the serum that react specifically to the coated HLA antigens are detected by using a FITC-conjugated antibody against human IgG . Percent PRA can be determined by the percentage of microbeads that react positively to the serum . There is no cross-reactivity between the Class I and Class II microbeads . A mixture of Class I and Class II microbeads can be distinguished by their different fluorescent properties on the flow cytometry analysis . Thus, both Class I and Class II PRA can be detected from a single tube reaction. Eur J Cancer, 1997 Dec, 33(14), 2384 - 9 Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells; Sathyamoorthy N et al.; The in vitro effects of two closely related phyto-oestrogens daidzein and equol on the oestrogen receptor positive human breast cancer cells MCF-7 were examined . There is differential metabolism of daidzein in humans, and the conversion of daidzein to equol by intestinal microbes occurs only in 30% of the population . The differential potency of these two compounds is thus of considerable importance since it may be likely that the relative risk of hormone-dependent cancers may be higher in 'non-responders' . In the present study, we compared the ability of both these compounds to induce mRNA expression of the oestrogen-responsive pS2 gene, to compete with oestradiol for binding to the oestrogen receptor (ER) and to affect cellular proliferation . Our studies demonstrate that equol is a 100-fold more potent than daidzein in stimulating an oestrogenic response . Equol was also more effective than daidzein in competing with 3H-oestradiol for binding to the ER . These results suggest that equol has a higher affinity for the ER . Both compounds stimulated the growth of MCF-7 cells in a concentration-dependent manner (10(-8)-10(-5)M) . Although equol exhibits oestrogenic activity, exposure of MCF-7 cells to equol simultaneously with oestradiol was effective in reducing pS2 mRNA expression . This was not observed with daidzein . However, long-term exposure of MCF-7 cells to both daidzein and equol resulted in the downregulation of ER mRNA expression. Am J Respir Cell Mol Biol, 1998 May, 18(5), 675 - 86 Ligation of the beta2 integrin triggers activation and degranulation of human eosinophils; Kato M et al.; Evidence suggests that cellular adhesion is critical for eosinophil effector functions . Here, we tested the hypothesis that an adhesion molecule, specifically beta2 integrin, participates in intracellular signaling events of eosinophils . Eosinophils stimulated with interleukin (IL)-5 and adherent to protein-coated tissue culture plates via beta2 integrin (CD18) showed tyrosine phosphorylation of a number of proteins . Among these proteins, tyrosine phosphorylation of the 105 kD and 115 kD proteins and the product of the c-cbl protooncogene, Cbl, was specifically inhibited using soluble anti-CD18 monoclonal antibody (mAb) to block eosinophil cell adhesion . Furthermore, phosphoinositide turnover of IL-5-stimulated adherent eosinophils was also inhibited by anti-CD18 mAb, suggesting that cellular adhesion plays important roles in eosinophil signal transduction . alphaM beta2 (Mac-1, CD11b/18) was one of the beta2 integrins involved in eosinophil adhesion to protein-coated plates . We found that direct ligation of eosinophil alphaM beta2 with anti-CD11b mAb coupled to polystyrene microbeads induced tyrosine phosphorylation of a 115 kD protein and Cbl . Furthermore, anti-CD11b mAb microbeads induced increases in both phosphoinositide hydrolysis and the eosinophil degranulation response . Control antibodies, such as mouse myeloma IgG1 and anti-HLA class I antigen mAb, did not induce these cellular responses . These results suggest that engagement of beta2 integrin either by cell adhesion or by anti-CD11b mAb triggers activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils . Tyrosine phosphorylation of a 115 kD protein and Cbl may play important roles in adhesion-dependent cellular functions of eosinophils. J Chromatogr B Biomed Sci Appl, 1998 Apr 10, 707(1-2), 25 - 31 Bilirubin removal from human plasma in a packed-bed column system with dye-affinity microbeads; Denizli A et al.; A dye-ligand . Cibacron Blue F3GA . was covalently coupled with the poly(EGDMA-HEMA) microbeads . The affinity sorbent carrying 16.5 micromol Cibacron Blue F3GA per gram polymer was then used to remove bilirubin from human plasma in a packed-bed column system . Bilirubin adsorption from human plasma on the unmodified poly(EGDMA-HEMA) microbeads was 0.32 mg/g, while much higher adsorption values, up to 24.2 mg/g, were obtained with the dye-attached microbeads . The bilirubin adsorption capacity of the microbeads decreased with an increase in the recirculation rate of plasma . Bilirubin adsorption increased with increasing temperature, and the maximum adsorption achieved at 37 degrees C (32.5 mg bilirubin/g polymer) . Bilirubin molecules interacted directly with the immobilized Cibacron Blue F3GA molecules . Contribution of albumin adsorption on bilirubin adsorption was also significant. J Nat Prod, 1998 May, 61(5), 591 - 7 Total synthesis and biological activities of (+)- and (-)-boscialin and their 1'-epimers; Busch J et al.; Natural (-)-boscialin {(-)-1} has recently been described as one of the constituents of various medicinal plants . To obtain more material for investigations of its biological activities, we carried out the synthesis of (-)-1 and its isomers . Starting from the chiral building block 2, the key steps of the synthesis involved a regioselective reduction and a nucleophilic addition . The enantiomer of the natural product, (+)-boscialin {(+)-1}, could be obtained via acid-catalyzed epimerization of hydroxyketone 4 to (+)-3 . Starting the synthesis with (-)-3 led to (-)-boscialin {(-)-1} with the natural absolute configuration . In addition to (+)- and (-)-boscialin, the corresponding 1'-epimers (+)- and (-)-epiboscialin were also obtained . In vitro assays with (-)-boscialin {(-)-1} and its three stereoisomers were carried out to test for activity against microbes, parasites, and human fibroblasts . The investigations revealed activity against various microbes and against Trypanosoma brucei rhodesiense and also revealed cytotoxicity against human cancer cells. Int J Oncol, 1998 Jun, 12(6), 1333 - 8 Detection of ras gene mutations in peripheral blood of carcinoma patients using CD45 immunomagnetic separation and nested mutant allele specific amplification; Shibata K et al.; We developed a sensitive technique of detecting circulating tumor cells in carcinoma patients, using CD45 immunomagnetic separation to isolate epithelial cells in blood samples and specific polymerase chain reaction analysis to identify point mutations of the K-ras gene . The method is based on the fact that the peripheral blood mononuclear cells (PBMC) that express CD45 antigen are trapped with anti-CD45 conjugated supramagnetic microbeads while the carcinoma cells that do not express CD45 antigen are not trapped and pass through the magnetic fields . This method concentrated the number of carcinoma cells 3.3 times . After this separation, the modified method of mutant allele specific amplification was applied and this method was able to ten control carcinoma cells in a background of 107 PBMC . A preliminary clinical study demonstrated that six cases of end-stage carcinoma with K-ras mutations in the primary tumor showed the same mutations in the peripheral blood samples, while two cases without K-ras mutation in the primary tumor and 10 healthy volunteers showed no mutation in the peripheral blood samples . The results suggest that this method may be very useful to detect circulating carcinoma cells in the patient whose primary tumor shows K-ras mutations. Rheum Dis Clin North Am, 1998 May, 24(2), 211 - 26 The arthritogenic properties of microbial antigens . Their implications in disease states; Zabriskie JB et al.; The sharing of antigenic determinants between host and microbe is a common event and new microbial-tissue cross-reactions are being recognized each year . Almost every human organ has been implicated as a possible target . The purpose of this article is to examine the arthritogenic properties of these microbial antigens and to explore the mechanisms by which they induce pathologic damage and disease. Antibiot Khimioter, 1998, 43(3), 27 - 30 {Pathomorphological changes in albino mice infected with plague and treated with cefotaxime}; Makarovskaia LN et al.; Pathomorphological and bacteriological changes in albino mice infected with plague and treated with cefotaxime were investigated . The control animals which died within 3 days had structural changes characteristic of generalized plague with lesions in the infection site, regional lymph nodes, spleen, liver and lungs . The plague microbe was isolated from the tissues of all the organs and blood . The animals treated with cefotaxime (200 mg/kg for 7 days) survived . The histological examination conclusively demonstrated the absence of the changes characteristic of generalized plague in their internal organs . The infection process was mainly restricted by the primary complex and was strictly localized . The tissue reaction around the focus in the second part of the experiment developed in accordance with the productive type inflammation followed by the organization and cicatrization . In the bacteriological investigation the plague causative agent was detectable during the first days of the treatment in the site of the infection . During the subsequent days the plague microbe was not detected. Neoplasma, 1997, 44(6), 348 - 55 Quantitative immunocytofluorometry--new parameters for the definition of leukemia cells; Babusikova O et al.; In our study we used for definition of leukemia/lymphoma cells a new parameter which allows the enumeration of mean fluorescence intensity expressed by the number of antigen molecules per cell . Quantitative immunofluorescence using calibration microbeads was performed in 36 patients with different acute and chronic lymphoid and myeloid leukemia and in 19 healthy volunteers . We showed that quantitative immunophenotyping allowed the definition of aberrant marker densities on neoplastic cells . We demonstrated under- and overexpression of CD8 marker in CD3/CD4/CD8 complex in T acute lymphatic leukemia and T non-Hodgkin's lymphoma and T leukemia of large granular lymphocytes as compared to normal counterparts . We pointed out that certain antigens (e . g . CD10, CD4, CD24) were expressed at different levels on different cell subsets (CD10 in early B-acute lymphatic leukemia and coexpressed in T-acute lymphatic leukemia, CD4 on T cells and monocytes, CD24 on B cells and granulocytes in chronic myeloid leukemia) . We showed that quantitative immune fluorescence could provide new data contributing to a more precise definition of cell differentiation . We documented the significant difference between antigen density of early and late markers in B-cell and myeloid malignancies . Further, we demonstrated that quantitative immune phenotyping could help in determination of exact definition of pathologic clone in morphologically immature leukemia population and showed that parameters of this method are also convenient for cytoplasmic marker evaluation . In our study we were able to demonstrate that CD45 quantitative expression appeared to be a more informative parameter than its percentage of antigen-positive cells as a measure of antigen expression only and we pointed out that low and high CD45 densities enabled to differentiate between pathological clone and residual healthy population in examined sample . We showed that quantitative immune phenotyping could be another important parameter for definition of leukemia phenotype suitable for detection of minimal residual disease. Biol Bull, 1998 Apr, 194(2), 116 - 9 Vestimentiferan on a whale fall; Feldman RA et al.; Discovery of chemosynthetic communities associated with whale bones led to the hypothesis that whale falls may serve as stepping-stones for faunal dispersal between disjunct hydrothermal vents and cold seeps on the ocean floor (1) . The initial observation was followed by a faunal inventory that revealed a diverse assemblage of microbes and invertebrates, supported by chemoautotrophic production, living in close proximity to whale remains (2, 3) . To date, the conspicuous absence from whale falls of vestimentiferan tubeworms (a predominant constituent of eastern Pacific vent and seep habitats) has been a major objection to the stepping-stone hypothesis (4-5) . We report the first evidence of a vestimentiferan tubeworm associated with a whale fall (Fig . 1) . The tubeworm, Escarpia spicata, was identified by morphological criteria and DNA sequence data from a portion of the mitochondrial cytochrome oxidase C subunit I (COI) gene . Additionally, the bacterial endosymbiont in the tubeworm possessed a 16S rRNA gene that was similar to that of endosymbionts from vestimentiferans in sedimented cold-seep environments. Arch Immunol Ther Exp (Warsz), 1997, 45(2-3), 189 - 94 Changes in the phagocytic cells in children treated with continuous ambulatory peritoneal dialysis; Wasik M et al.; Children on continuous ambulatory peritoneal dialysis (CAPD) in endstage renal failure are highly exposed to peritonitis . Peritoneal macrophages (PM) and blood neutrophils (PMNC) are the first line of defense against invading microbes . This study was undertaken for assessing surface receptors expression on PM and PMNC and to check their ability to phagocytosis and killing of bacteria . We have found that in spite of the decreased number of PM in dialysate fluid their viability and activity significantly increased during CAPD . Moreover, higher number of PM expressed CD16 and CD35 antigens (FcRIII and C3bR, respectively) in comparison with the results observed at CAPD onset . The number of PMNC expressed of these two antigens in uremic children blood were significantly lower in comparison with healthy control . The number of CD16 positive cells increased under influence of CAPD only temporarily . CAPD caused improvement of phagocytosis and intracellular killing of bacteria by PM but not by PMNC . There is discussed here influence of uremia and CAPD on surface antigens, function of phagocytes as well as renewal of PM during CAPD. Acta Vet Scand, 1998, 39(1), 25 - 33 Temporary suppression of cell-mediated immunity in standardbred horses with decreased athletic capacity; Jensen-Waern M et al.; Eighty Standardbred horses, originating from 5 training campuses, with decreased athletic performance in association with symptoms such as intermittent fever and mild pharyngitis were examined . As control animals, 10 horses from a stable with normally performing horses were used . Virus isolation and clinico-chemical and serological tests were performed . Lymphocyte proliferation tests were carried out to evaluate the capacity of the cell-mediated immunity . In addition, a bioassay for equine type I interferon, as a marker for early viral infections, was established . No specific microbe could be linked to these symptoms, but there was a temporary suppression of the cell-mediated immunity, which might be explained by the serological evidence of an EHV-2 and/or rhinovirus infection. Acta Pharm Hung, 1998 Mar, 68(2), 127 - 32 {Filtration of solutions with high viscosity: from laboratory experiments to production}; Bodis A et al.; The Zinc Hyaluronate complex has several advantages from the point of view of woundhealing . The author's task was the elaboration of the sterile filtration technology of the 0.2% Zinc Hyaluronate solution . In the first step 0.2 micron pore diameter filters were used . During the scale up process we found that the filter boards are not suitable . The filtration time extremely increased and sometimes the whole process stopped because of the relatively small filtration area . For larger batches polypropylene capsule filters have been applied . The mean pore diameter of the filter was 0.2 micron but the unregular pore size distribution did not make the filtrate microbe free . In the next experience inlet pore diameter was 0.65 micron--the outlet pore diameter 0.45 micron . This filtration process resulted in a sterile filtrate with reduced active content . This means that the solution has been ultrafiltrated . Applying too high pressure and form a compact layer on the filter's surface which is functioning as a secondary filter layer . The filtration should begin with low pressure and it has to increase it gradually from 0 to 2 bar in 0.5 bar steps . It was supposed that at high temperature filtration could be easier and faster . On the contrary we found that depending on the concentration above 35-45 degrees C the active content of the filtrate is decreasing . In the case of the filtration of the fraction with a lower temperature the fractions with high molecular weight can be filtrated because of the anisometric distribution of the molecules . The filtration of these products is very difficult even at laboratory scale. Biophys J, 1998 May, 74(5), 2689 - 701 Separation of polystyrene microbeads using dielectrophoretic/gravitational field-flow-fractionation; Wang XB et al.; The characterization of a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system using model polystyrene (PS) microbeads is presented . Separations of PS beads of different surface functionalization (COOH and none) and different sizes (6, 10, and 15 microm in diameter) are demonstrated . To investigate the factors influencing separation performance, particle elution times were determined as a function of particle suspension conductivity, fluid flow rate, and applied field frequency and voltage . Experimental data were analyzed using a previously reported theoretical model and good agreement between theory and experiment was found . It was shown that separation of PS beads was based on the differences in their effective dielectric properties . Particles possessing different dielectric properties were positioned at different heights in a fluid-flow profile in a thin chamber by the balance of DEP and gravitational forces, transported at different velocities under the influence of the fluid flow, and thereby separated . To explore hydrodynamic (HD) lift effects, velocities of PS beads were determined as a function of fluid flow rate in the separation chamber when no DEP field was applied . In this case, particle equilibrium height positions were governed solely by the balance of HD lift and gravitational forces . It was concluded that under the experimental conditions reported here, the DEP force was the dominant factor in controlling particle equilibrium height and that HD lift force played little role in DEP/G-FFF operation . Finally, the influence of various experimental parameters on separation performance was discussed for the optimization of DEP/G-FFF. Electrophoresis, 1998 Apr, 19(4), 608 - 12 Whole genome scan for habitat-specific genes by signature-tagged mutagenesis; Hensel M; Large numbers of new open reading frames can be identified by whole genome sequencing of microbial genomes . Efficient new approaches are required to investigate the role of the putative genes, or to identify genes required in distinct habi