Mol Microbiol, 2004 Jun, 52(5), 1243 - 53 Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences; Lovett ST; Mutations and rearrangements that occur by misalignment during DNA replication are frequent sources of genetic variation in bacteria . Dislocations between a replicating strand and its template at repetitive DNA sequences underlie the mechanism of these genetic events . Such misalignments can be transient or stable and can involve intramolecular or intermolecular DNA mispairing, even pairing across a replication fork . Paradoxically, these replication 'slippage' events both create and destroy repetitive sequences in bacterial genomes . This review catalogues several types of slippage errors, presents the cellular processes that act to limit them and discusses the consequences of this class of genetic events on the evolution of bacterial genomes and physiology. J AOAC Int, 2004 Mar-Apr, 87(2), 545 - 62 Methods for analysis of conjugated linoleic acids and trans-18:1 isomers in dairy fats by using a combination of gas chromatography, silver-ion thin-layer chromatography/gas chromatography, and silver-ion liquid chromatography; Cruz-Hernandez C et al.; Conjugated linoleic acids (CLA) are octadecadienoic acids (18:2) that have a conjugated double-bond system . Interest in these compounds has expanded since CLA were found to be associated with a number of physiological and pathological responses such as cancer, metastases, atherosclerosis, diabetes, immunity, and body fat/protein composition . The main sources of these conjugated fatty acids are dairy fats . Rumen bacteria convert polyunsaturated fatty acids, especially linoleic and linolenic acids, to CLA and numerous trans- containing mono- and diunsaturated fatty acids . It has been established that an additional route of CLA synthesis in ruminants and monogastric animals, including humans, occurs via delta9 desaturation of the trans-18:1 isomers . To date, a total of 6 positional CLA isomers have been found in dairy fats, each occurring in 4 geometric forms (cis,trans; trans,cis; cis,cis; and trans,trans) for a total of 24 . All of these CLA isomers can be resolved only by a combination of gas chromatography (GC), using 100 m highly polar capillary columns, and silver-ion liquid chromatography, using 3 of these 25 cm columns in series . Complete analysis of all the trans-18:1 isomers requires prior isolation of trans monoenes by silver-ion thin-layer chromatography (TLC), followed by GC analysis using the same 100 m capillary columns operated at low temperatures starting from 120 degrees C . These analytical techniques are required to assess the purity of commercial CLA preparations, because their purity will affect the interpretation of any physiological and/or biochemical response obtained . Prior assessment of CLA preparations by TLC is also recommended to determine the presence of any other impurities . The availability of pure CLA isomers will permit the evaluation and analysis of individual CLA isomers for their nutritional and biological activity in model systems, animals, and humans . These techniques are also essential to evaluate dairy fats for their content of specific CLA isomers and to help design experimental diets to increase the level of the desired CLA isomers in dairy fats . These improved techniques are further required to evaluate the CLA profile in monogastric animals fed commercial CLA preparations for CLA enrichment of animal products . This is particularly important because absorption and metabolism will alter the ingested-CLA profile in the animal fed. Trop Gastroenterol, 2003 Oct-Dec, 24(4), 185 - 8 Association of human leucocyte DR and DQ antigens in Crohn's disease in Asian Indians: a family study; Thakur S et al.; The pathogenesis of Crohn's disease (CD) involves an abnormal immune response to enteric bacteria in genetically susceptible individuals . There are no family studies regarding the association of CD with human leucocyte antigens (HLA) class II . In the present study, we have studied the association of HLA class II antigens in patients with CD and their first-degree relatives . Nine patients with CD and their first-degree relatives were studied . A group of 110 healthy unrelated and ethnically matched subjects were used as controls . Molecular HLA typing was done using the sequence-specific primer-based method . The transmission disequilibrium test (TDT) was used to analyze the results . A total of 65 individuals were included in the study; 52/56 first-degree relatives (92.8%) of 9 patients with CD consented to the study . The median age of patients was 40 years . When the distribution of the HLA class II antigens in patients was compared to that in controls no significant differences were observed even after applying the Yates correction . As the sample size of the population was small, the association of CD with DR and DQ alleles was further analyzed by using the TDT . Even after applying TDT, no significant association was observed . Familial aggregation of CD is uncommon in India . Crohn disease is not associated with HLA class II antigens in Indian patients . Genes of the major histocompatiblity complex are likely to contribute little to the susceptibility to Crohn disease in Indian patients. Microb Ecol, 2004 Jul, 48(1), 51 - 65 Epub 2004 May 06. Plankton diversity in the Bay of Fundy as measured by morphological and molecular methods; Savin MC et al.; Phytoplankton have traditionally been identified based on morphological characteristics . However, identifications based on morphology are time-consuming, require expertise in taxonomy, and often fail to distinguish differences among the multitudes of minute, nondescript planktonic organisms . Molecular techniques, which have revealed new insights into bacterial and picoplankton communities, may also enhance our knowledge of the diversity among communities of larger plankton . We compared plankton identifications and community assessments based on the two types of techniques (morphological vs molecular) for surface seawater samples collected on 2 May, 31 July and 25 September 2000 from several sampling stations in the Bay of Fundy . Phytoplankton captured in surface bucket samples were quantified and identified based on morphology . DNA was extracted from plankton communities (5-100 microm in diameter) collected by filtration, and 18S rRNA gene fragments were amplified with primers specific for eukaryotes . Denaturing gradient gel electrophoresis (DGGE) was used to develop DNA profiles of eukaryotic phylogenetic diversity and to select cloned 18S rDNA fragments for sequencing . Both morphological and molecular methods showed great community diversity . However, the communities identified with the two different types of techniques were starkly different . Morphological abundances and taxon richness were lowest in the May samples, whereas the number of DGGE bands was highest in May and July . Morphological identifications showed a succession of dominant organisms through time . Whereas neither diatoms nor dinoflagellates were dominant in May, diatoms and a few dinoflagellates were dominant in July and September . In contrast, few 18S rDNA sequences were related to rDNA sequences of known identity, and furthermore, few diatoms were identified in the molecular analyses . Molecular phylogenetic analysis indicated the presence of many novel organisms, several of which were most closely related to other unidentified sequences from diverse marine environments representing new lineages . Our results support the ideas that we are just beginning to uncover the diversity of eukaryotic marine organisms and that there may be many more ubiquitous, microeukaryotic plankton than previously realized . Our results suggest that both types of methods capture only a portion of the community . Morphological methods may be more adept at capturing the phototrophic organisms within the community . However, just as for bacteria and picoplankton, molecular techniques can enhance our understanding of plankton diversity, particularly by detecting previously unidentified organisms. Mol Biol Evol, 2004 Sep, 21(9), 1692 - 703 Epub 2004 May 26. The cation/Ca(2+) exchanger superfamily: phylogenetic analysis and structural implications; Cai X et al.; Cation/Ca(2+) exchangers are an essential component of Ca(2+) signaling pathways and function to transport cytosolic Ca(2+) across membranes against its electrochemical gradient by utilizing the downhill gradients of other cation species such as H(+), Na(+), or K(+) . The cation/Ca(2+) exchanger superfamily is composed of H(+)/Ca(2+) exchangers and Na(+)/Ca(2+) exchangers, which have been investigated extensively in both plant cells and animal cells . Recently, information from completely sequenced genomes of bacteria, archaea, and eukaryotes has revealed the presence of genes that encode homologues of cation/Ca(2+) exchangers in many organisms in which the role of these exchangers has not been clearly demonstrated . In this study, we report a comprehensive sequence alignment and the first phylogenetic analysis of the cation/Ca(2+) exchanger superfamily of 147 sequences . The results present a framework for structure-function relationships of cation/Ca(2+) exchangers, suggesting unique signature motifs of conserved residues that may underlie divergent functional properties . Construction of a phylogenetic tree with inclusion of cation/Ca(2+) exchangers with known functional properties defines five protein families and the evolutionary relationships between the members . Based on this analysis, the cation/Ca(2+) exchanger superfamily is classified into the YRBG, CAX, NCX, and NCKX families and a newly recognized family, designated CCX . These findings will provide guides for future studies concerning structures, functions, and evolutionary origins of the cation/Ca(2+) exchangers. J Virol, 2004 Jun, 78(12), 6459 - 68 Adenovirus protein VII condenses DNA, represses transcription, and associates with transcriptional activator E1A; Johnson JS et al.; Adenovirus protein VII is the major protein component of the viral nucleoprotein core . It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex . We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII . Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes . Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes . Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes . During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein . To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes . Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes . Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro . Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII . These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes. Mol Immunol, 2004 Jun, 41(4), 355 - 67 The human complement factor H: functional roles, genetic variations and disease associations; Rodriguez de Cordoba S et al.; Factor H is an essential regulatory protein that plays a critical role in the homeostasis of the complement system in plasma and in the protection of bystander host cells and tissues from damage by complement activation . Genetic and structural data generated during recent years have been instrumental to delineate the functional domains responsible for these regulatory activities in factor H, which is helping to understand the molecular basis underlying the different pathologies associated to factor H . This review summarises our current knowledge of the role of factor H in health and disease. Cell, 2004 May 28, 117(5), 677 - 87 Polysaccharide processing and presentation by the MHCII pathway; Cobb BA et al.; The adaptive immune system functions through the combined action of antigen-presenting cells (APCs) and T cells . Specifically, class I major histocompatibility complex antigen presentation to CD8(+) T cells is limited to proteosome-generated peptides from intracellular pathogens while the class II (MHCII) endocytic pathway presents only proteolytic peptides from extracellular pathogens to CD4(+) T cells . Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria can activate CD4(+) T cells . Here we show that ZPSs are processed to low molecular weight carbohydrates by a nitric oxide-mediated mechanism and presented to T cells through the MHCII endocytic pathway . Furthermore, these carbohydrates bind to MHCII inside APCs for presentation to T cells . Our observations begin to elucidate the mechanisms by which some carbohydrates induce important immunologic responses through T cell activation, suggesting a fundamental shift in the MHCII presentation paradigm. Ageing Res Rev, 2004 Jan, 3(1), 105 - 20 Infectious agents and age-related neurodegenerative disorders; Mattson MP; chlamdAs with other organ systems, the vulnerability of the nervous system to infectious agents increases with aging . Several different infectious agents can cause neurodegenerative conditions, with prominent examples being human immunodeficiency virus (HIV-1) dementia and prion disorders . Such infections of the central nervous system (CNS) typically have a relatively long incubation period and a chronic progressive course, and are therefore increasing in frequency as more people live longer . Infectious agents may enter the central nervous system in infected migratory macrophages, by transcytosis across blood-brain barrier cells or by intraneuronal transfer from peripheral nerves . Synapses and lipid rafts are important sites at which infectious agents may enter neurons and/or exert their cytotoxic effects . Recent findings suggest the possibility that infectious agents may increase the risk of common age-related neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and stroke . While scenarios can be envisioned whereby viruses such as Chlamydia pneumoniae, herpes simplex and influenza promote damage to neurons during aging, there is no conclusive evidence for a major role of these pathogens in neurodegenerative disorders . In the case of stroke, blood vessels may be adversely affected by bacteria or viruses resulting in atherosclerosis. Int J Toxicol, 2004, 23 Suppl 1, 29 - 57 Safety assessment of MIBK (methyl isobutyl ketone); Johnson W Jr; MIBK (Methyl Isobutyl Ketone) is an aliphatic ketone that functions as both a denaturant and solvent in cosmetic products . Current use in cosmetic products is very limited, but MIBK is reported to be used in one nail correction pen (volume = 3 ml) at a concentration of 21% . The maximum percutaneous absorption rate in guinea pigs is 1.1 micromol/min/cm2 at 10 to 45 min . Metabolites include 4-hydroxy-4-methyl-2-pentanone (oxidation product) and 4-methyl-2-pentanol (4-MPOL) (reduction product) . Values for the serum half-life and total clearance time of MIBK in animals were 66 min and 6 h, respectively . In clinical tests, most of the absorbed MIBK had been eliminated from the body 90 min post exposure . MIBK was not toxic via the oral or dermal route of exposure in acute, short-term, or subchronic animal studies, except that nephrotoxicity was observed in rats dosed with 1 g/kg in a short-term study . MIBK was an ocular and skin irritant in animal tests . Ocular irritation was noted in 12 volunteers exposed to 200 ppm MIBK for 15 min in a clinical test . A depression of the vestibulo-oculomotor reflex was seen with intravenous infusion of MIBK (in an emulsion) at 30 microM/kg/min in female rats . The no-observed-effect level in rats exposed orally to MIBK was 50 mg/kg . Both gross and microscopic evidence of lung damage were reported in acute inhalation toxicity studies in animals . Short-term and subchronic inhalation exposures (as low as 100 ppm) produced effects in the kidney and liver that were species and sex dependent . Dermal doses of 300 or 600 mg/kg for 4 months in rats produced reduced mitotic activity in hair follicles, increased thickness of horny and granular cell layers of the epidermis, a decrease in the number of reactive centers in follicles (spleen), an increase in the number of iron-containing pigments in the area of the red pulp (spleen), and a reduction in the lipid content of the cortical layer of the adrenal glands . Neuropathological changes in the most distal portions of the tibial and ulnar nerves were observed in young adult rats which inhaled 1500 ppm MIBK for up to 5 months . No adverse effects were seen in any other neurological end point by any route of exposure in other studies using rats or other animal species . Clinical tests demonstrated a threshold for MIBK-induced irritation of the lungs at 0.03 to 0.1 mg/L after 1 min of respiration . MIBK was not mutagenic in the Ames test or in a mitotic gene-conversion assay in bacteria . Mammalian mutagenicity test results were also negative in the following assays: mouse lymphoma, unscheduled DNA synthesis, micronucleus, cell transformation, and chromosome damage . MIBK did not induce any treatment-related increases in embryotoxicity or fetal malformations in pregnant Fischer 344 rats or CD-1 mice that inhaled MIBK at concentrations of 300, 1000, or 3000 ppm . There was evidence of treatment-related maternal toxicity only at the highest concentration tested . MIBK applied to the tail of rats daily at doses of 300 or 600 mg/kg for 4 months produced changes in the testes, including a reduction in the number of spermatocytes, spermatids, and spermatozoa . An ongoing carcinogenicity study of MIBK being conducted by the National Toxicology Program will be considered when the results are available . On the basis of the information that is currently available, MIBK is considered safe as used in nail polish removers and as an alcohol denaturant in cosmetic products. Proteins, 2004 Jul 1, 56(1), 19 - 27 Predicted role for the archease protein family based on structural and sequence analysis of TM1083 and MTH1598, two proteins structurally characterized through structural genomics efforts; Canaves JM; Recently, the structures of two proteins belonging to the archease family, TM1083 from Thermotoga maritima and MTH1598 from Methanobacterium thermoautotrophicum, have been solved independently by two Protein Structure Initiative structural genomics pilot centers using X-ray crystallography and NMR, respectively . The archease protein family is a good example of one of the paradoxes of structural genomics: Approximately one third of protein structures produced by structural genomics centers have no known function and are still annotated as "hypothetical proteins" in the Protein Data Bank . In the case of archeases, despite the existence of two protein structures and abundant sequence information, there is still no function assigned to this protein family . Here, our group predicts, based on structural similarity, sequence conservation, and gene context analyses, that members of this protein family might function as chaperones or modulators of proteins involved in DNA/RNA processing . The conservation of genomic context for this protein family is constant from Archaea and Bacteria to humans, and suggests that unannotated open reading frames contiguous to them could be novel RNA/DNA binding proteins . Biotechnol Bioeng, 2004 Jun 30, 86(7), 775 - 87 Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration; Guarino RD et al.; We describe a simple protocol for determining the oxygen consumption of cells in static culture . The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law . The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension . Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen . The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model . Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic . This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero . Such determinations were performed for numerous cell types, in varying well sizes . Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes . This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture . Cytogenet Genome Res, 2004, 104(1-4), 7 - 13 Pathways of DNA double-strand break repair and their impact on the prevention and formation of chromosomal aberrations; Pfeiffer P et al.; DNA double-strand breaks (DSB) are considered the critical primary lesion in the formation of chromosomal aberrations (CA) . DSB occur spontaneously during the cell cycle and are induced by a variety of exogenous agents such as ionising radiation . To combat this potentially lethal damage, two related repair pathways, namely homologous recombination (HR) and non-homologous DNA end joining (NHEJ), have evolved, both of which are well conserved from bacteria to humans . Depending on the pathway used, the underlying mechanisms are capable of eliminating DSB without alterations to the original genomic sequence (error-free) but also may induce small scale mutations (base pair substitutions, deletions and/or insertions) and gross CA (error-prone) . In this paper, we review the major pathways of DSB-repair, the proteins involved therein and their impact on the prevention of CA formation and carcinogenesis . Invest Ophthalmol Vis Sci, 2004 Jun, 45(6), 1804 - 11 TAT-mediated protein transduction into human corneal epithelial cells: p15(INK4b) inhibits cell proliferation and stimulates cell migration; Guo X et al.; PURPOSE: The cell cycle inhibitor p15(INK4b) has been localized in migrating corneal epithelial cells . In this study, TAT-fusion protein technology was used to transduce p15(INK4b) into human corneal epithelial cells to examine the effect on cell proliferation and migration . METHODS: Human p15(INK4b), obtained by RT-PCR, was cloned into a TAT-HA vector, and the fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct . Various dilutions of TAT-HA-p15 were applied to primary human corneal epithelial cells to test potency . In addition, the effect of exposure time was examined . Cells were labeled with bromodeoxyuridine to detect proliferation, and indirect immunofluorescence was performed . Ki67 expression was also examined . To assay cell migration, human corneal epithelial cells were plated inside a cylinder and exposed to TAT-HA-p15 . The cylinder was removed, the cells were allowed to spread for 2 days, and the area of cell coverage was calculated . TAT-HA-beta-galactosidase served as the control in all experiments . Finally, the extent of retinoblastoma protein phosphorylation was assayed by Western blot in cells cultured with and without TAT-HA-p15 . RESULTS: TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells . TAT-HA-p15 decreased proliferation in a concentration- and time-dependent manner . The migration assay showed that TAT-HA-p15 stimulated cell migration 1.8-fold . TAT-HA-beta-galactosidase had no effect on proliferation or migration . Finally, TAT-HA-p15 decreased the level of phosphorylated retinoblastoma protein by 4.9-fold . CONCLUSIONS: Active p15(INK4b) can be efficiently transduced into primary human corneal epithelial cells using TAT-fusion protein technology . p15(INK4b) appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration. J Environ Manage, 2004 Feb, 70(2), 101 - 7 Impact of food industrial waste on anaerobic co-digestion of sewage sludge and pig manure; Murto M et al.; The performance of an anaerobic digestion process is much dependent on the type and the composition of the material to be digested . The effects on the degradation process of co-digesting different types of waste were examined in two laboratory-scale studies . In the first investigation, sewage sludge was co-digested with industrial waste from potato processing . The co-digestion resulted in a low buffered system and when the fraction of starch-rich waste was increased, the result was a more sensitive process, with process overload occurring at a lower organic loading rate (OLR) . In the second investigation, pig manure, slaughterhouse waste, vegetable waste and various kinds of industrial waste were digested . This resulted in a highly buffered system as the manure contributed to high amounts of ammonia . However, it is important to note that ammonia might be toxic to the micro-organisms . Although the conversion of volatile fatty acids was incomplete the processes worked well with high gas yields, 0.8-1.0 m3 kg(-1) VS. Shanghai Kou Qiang Yi Xue, 1994 Jun, 3(2), 82 - 4 {Neutrophils and monocytes in gingival epithelium}; Meng HX et al.; Neutrophils and monocytes of gingival epithellium in health gingiva(H),marginal gingivitis(MG),juvenile periodontitis(JP),adult periodontitis(AP) and subgingival bacteria were quantitated and analyzed,The results showed that the numbers of PMN within either pocket epithelium or oral gingival epithelium in JP were significantly lower than in AP and G.The amounts of PMN in AP were much larger than other three groups.Positive correlation between the number of PMN in sulcular pocket epitelium and the motile bacteri of subgingival plaque was demonstrated by correlation analysis.Monocytes mainly presented in deep pocket and junctional epithelum which were stained by NAE method,however very few Langhans cells were seen in these areas. Shanghai Kou Qiang Yi Xue, 1993 Jun, 2(2), 63 - 6 {Ductal exfoliated cytology in the practice of mumps}; Bao XH; The author used smear from Stensen's duct aspiration to observe exfoliated cytology in order to help diagnosis of early mumps.According to the smears from 100 cases of mumps and other 50 cases of bacteria parotitis and/or normal parotid as contro,the author revealed that if there are a lot of ductal epithelial cells,monocytes and polynuclear cells,monocytes and polynuclear phagocytes at same time,it could give diagnosis "Mumps" to that patient.In control group,it is unable to observe those three kind cells at same time.The author also discussed the mechanism of how would these three kind cells appear in mumps in accordance with references. Plant Mol Biol, 2004 Jan, 54(1), 39 - 54 A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers; Gray J et al.; Conservation of Lethal-leaf spot 1 (Lls1) lesion mimic gene in land plants including moss is consistent with its recently reported function as pheophorbide a oxygenase (Pao) which catalyzes a key step in chlorophyll degradation (Pruzinska et al., 2003) . A bioinformatics survey of complete plant genomes reveals that LLS1(PAO) belongs to a small 5-member family of non-heme oxygenases defined by the presence of Rieske and mononuclear iron-binding domains . This gene family includes chlorophyll a oxygenase (Cao), choline monooxygenase (Cmo), the gene for a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic 55) and a novel 52 kDa protein isolated from chloroplasts (Ptc 52) . Analysis of gene structure reveals that these genes diverged prior to monocot/dicot divergence . Homologues of LLS1(PAO), CAO, TIC55 and PTC52 but not CMO are found in the genomes of several cyanobacteria . LLS1(PAO), PTC52, TIC55 and a set of related cyanobacterial homologues share an extended carboxyl terminus containing a novel F/Y/W-x(2)-H-x(3)-C-x(2)-C motif not present in CAO . These proteins appear to have evolved during the transition to oxygenic photosynthesis to play various roles in chlorophyll metabolism . In contrast, CMO homologues are found only in plants and are most closely related to aromatic ring-hydroxylating enzymes from soil-dwelling bacteria, suggesting a more recent evolution of this enzyme, possibly by horizontal gene transfer . Our phylogenetic analysis of 95 extant non-heme dioxygenases provides a useful framework for the classification of LLS1(PAO)-related non-heme oxygenases. Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1137 - 8 Epub 2004 May 21. Expression, purification and preliminary X-ray diffraction analysis of a ketoreductase from a type II polyketide synthase; Teartasin W et al.; Polyketide metabolites produced by bacteria and other organisms include antibiotics, anticancer and antifungal compounds . In type II polyketide synthesis, three enzymes are sufficient to form a polyketide product of the requisite chain length, although the fidelity of the first cyclization is variable . Addition of ketoreductase (KR) to this system results in the formation of a product with correct cyclization and reduction . This paper reports the cloning of the Streptomyces coelicolor actIII ORF5 gene that codes for the ketoreductase . The 261-amino-acid protein has been overexpressed with a 20-residue His tag, purified by affinity chromatography and crystallized in space group P3(2)21, with unit-cell parameters a = b = 103.9, c = 123.1 angstroms . The crystals diffract to 2.5 angstroms resolution . A complete data set has been collected and structure solution and refinement is under way . J Biol Chem, 2004 Aug 6, 279(32), 33623 - 9 Epub 2004 May 24. Oligomerization of the sensory and motor neuron-derived factor prevents protein O-glycosylation; Cabedo H et al.; The sensory and motor neuron-derived factor (SMDF) is a neuregulin that promotes Schwann cell proliferation and differentiation . Hence, understanding axon myelination is important to unveil the mechanisms involved in SMDF biogenesis, membrane delivery, and compartmentalization . SMDF is a type II membrane protein expressed as two distinct polypeptides of approximately 40 and 83 kDa . Whether the 83-kDa polypeptide results from posttranslational modifications of the protein monomers or protein dimerization remains unknown . Here we have addressed this question and shown that the 83-kDa polypeptide is an O-glycosylated form of the protein . Deletion of the N-terminal domain fully abrogates the SMDF O-glycosylation, indicating that incorporation of O-glycans occurs in the intracellular domain of the protein . Notably, O-glycosylated forms are excluded from partitioning into lipid raft microdomains . In addition, we found that heterologously expressed SMDF monomers interact in intact living cells as evidenced from fluorescence resonance energy transfer of cyan fluorescent protein/yellow fluorescent protein.SMDF fusion proteins . A stepwise deletion approach demonstrated that SMDF self-association is primarily determined by its transmembrane segment . Notably, biochemical analysis revealed that SMDF multimers are exclusively composed of the 40-kDa polypeptide . Collectively, these findings indicate that the 40-kDa form corresponds to unmodified SMDF, which may be present as multimers, whereas the 83-kDa polypeptide is a monomeric O-glycosylated form of the protein . Furthermore, our observations imply a role for oligomerization as a potential modulator of the distribution in membrane domains and O-glycosylation of the protein. Biomaterials, 2004 Nov, 25(25), 5593 - 601 Biocompatibility of poly(epsilon-caprolactone)/poly(ethylene glycol) diblock copolymers with nanophase separation; Hsu SH et al.; In this study, we prepared diblock copolymers of poly(epsilon-caprolactone) (PCL) and poly(ethylene glycol) (PEG) by aluminum alkoxide catalysts . The biological responses to the spin cast surface of different PCL/PEG diblock copolymers were investigated in vitro . Our results showed that surface hydrophilicity improved with the increased PEG segments in diblock copolymers and that bacteria adhesion was inhibited by increased PEG contents . PCL-PEG 23:77 showed nanotopography on the surface . The number of adhered endothelial cells, platelets and monocytes on diblock copolymer surfaces was inhibited in PCL-PEG 77:23 and enhanced in PCL-PEG 23:77 . Nevertheless, the platelet and monocyte activation on PCL-PEG 23:77 was reduced . PCL-PEG 23:77 had better cellular response as well as lower degree of platelet and monocyte activation . The current study was the first one to demonstrate that surface nanotopography could influence not only cell adhesion and growth but also platelet and monocyte activation. Fitoterapia, 2004 Jun, 75(3-4), 405 - 8 Biological screening of Bangladeshi mango mistletoe bark extracts; Islam R et al.; The ethyl acetate extract of the Bangladeshi mango mistletoe (Loranthus globosus) bark was found to be most effective against both Gram (+) and Gram (-) bacteria and it also showed good cytotoxicity with a LC50 10.83 microg/ml . Biochim Biophys Acta, 2004 Jun 1, 1699(1-2), 87 - 94 Saturation mutagenesis, complement selection, and steady-state kinetic studies illuminate the roles of invariant residues in active site loop I of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi; Butterworth AC et al.; Hypoxanthine phosphoribosyltransferases (HPRTs) are potential drug targets in the treatment of diseases caused by parasites . Also, defects in the human HPRT can result in gouty arthritis or Lesch-Nyhan syndrome . Active site loop I of HPRTs has been implicated in interactions between enzyme subunits that can influence the relative efficiencies of forward and reverse reactions, but the functional roles for invariant loop I residues (analogous with human Leu67 and Gly69) are poorly understood . Herein, saturation mutagenesis, complement selection, and steady-state kinetics were used to investigate the functional roles for Leu67 and Gly69 . Seventy clones from a library of mutants were sequenced and more than 30 different mutations, or combinations of mutations, were identified . Several recombinant HPRTs with mutations at positions 67 and/or 69 supported the growth of a bacterial auxotroph on selective media, but only two of the mutants (L67M and G69S) could be recovered in the soluble fraction from bacteria induced to over-express the enzyme . The results of steady-state kinetic studies for L67M are consistent with the side chain of this residue participating in hydrophobic interactions between dimer subunits that are important for the proper positioning of main chain atoms that influence enzyme chemistry and the binding of PRPP, PPi, and hypoxanthine . The results for mutations at position 69 are consistent with only hydrogen or a small polar side chain being tolerated at this site . Kinetic studies of G69S suggest that side chains of residues at position 69 that project into the active site likely interfere with the binding of PRPP and PPi, as well as the positioning of a metal ion that indirectly influences the binding of purine bases and purine moieties of nucleotide substrates. Bioresour Technol, 2004 Sep, 94(2), 137 - 42 Survival and viability of Ascaris suum and Oesophagostomum dentatum in ensiled swine faeces; Caballero-Hernandez AI et al.; The survival and viability of eggs from Ascaris suum and Oesophagostomum dentatum and of infective larvae (L3) from O . dentatum were determined in the ensiled solid fraction of swine faeces after 0, 7, 14, 28 and 56 days of ensiling . The experiment had two treatments, un-ensiled and ensiled manure, in a split-plot design . Each of 50 containers was inoculated with 40,000 eggs of both A . suum and O . dentatum, and another 50 containers were inoculated with 32,747 L3 of O . dentatum each . A . suum eggs were not destroyed by the ensiling process, although their viability was diminished . O . dentatum eggs and larvae were destroyed during the first 7-14 days of the ensiling process . Anal Biochem, 2004 Jun 15, 329(2), 230 - 7 Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase; Otsuji T et al.; Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis . We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes . The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal ( {Formula: see text} nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET) . The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells . NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions . Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion . The change in BRET signals quantifies the processing of the fusion protein . Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells. J Colloid Interface Sci, 2004 Jul 1, 275(1), 82 - 9 Cadmium complexation by bacteriogenic iron oxides from a subterranean environment; Martinez RE et al.; This study quantifies the metal sorption characteristics of subterranean bacteriogenic iron oxides (BIOS) and their organic phases (intermixed intact and fragmented bacteria) . A Cd2+ ion-selective electrode was used to generate high-resolution metal sorption data as a function of increasing pH . A multisite Langmuir model, along with a linear programming regression method (LPM), was applied to fit experimental data . This approach found two discrete Cd2+ binding sites for the BIOS with average -log10 equilibrium constants (pK(S,j)) of 1.06 +/- 0.19 and 2.24 +/- 0.28 . Three discrete sites were obtained for the bacterial fraction, with pK(S,j) values of -0.05 +/- 0.12, 1.18 +/- 0.02, and 3.81 +/- 0.16 . This indicated that the BIOS surface had a lower affinity for Cd2+ than that of the bacteria . pK(S,j) values for the BIOS were similar to those reported for pure iron oxide phases, while the organic fraction pK(S,j) spectrum was consistent with previous spectra for intact bacteria . Individual binding site densities of 0.04 +/- 0.01 and 0.05 +/- 0.02 and 0.29 +/- 0.05, 0.11 +/- 0.01, and 0.09 +/- 0.02 micromol/mg of BIOS corresponded to the iron oxide mixture and bacteria fraction, respectively . These values indicated high concentrations of strong affinity Cd2+ complexing groups on the bacterial surface . Comparison of total site densities of 0.08 +/- 0.02 and 0.48 +/- 0.06 micromol/mg of BIOS for the mixture and the bacterial phase, respectively, suggested a nonadditive character for the BIOS surface reactivity . This was emphasized by a higher affinity for Cd2+, as well as an increase in total site concentration observed for the bacterial phase . LPM was able to distinguish between the BIOS mixture and its organic fraction Cd2+ complexation characteristics . This approach is therefore a useful tool for the study of natural sorbent materials controlling metal partitioning in contaminated and pristine environments. Best Pract Res Clin Gastroenterol, 2004 Jun, 18(3), 451 - 62 Inflammatory bowel disease: a complex group of genetic disorders; Hugot JP; Inflammatory bowel disease (IBD) are complex genetic disorders resulting from the complex interplay between several genetic and environmental risk factors . The number of IBD genes is currently unknown but it is expected to be equal or higher to 8 . None of these genes are expected to be neither necessary nor sufficient for disease development . Among the candidate genes investigated to date, only CARD15/NOD2 has been definitively associated with Crohn Disease (CD) but not Ulcerative Colitis (UC) . This gene explains about 20% of the genetic predisposition to CD . Because it is involved in the innate immunity, it allows to speculate that CD is the result of a defect in host-bacteria interaction . However, it is not known if CD is caused by specific bacteria or the gut flora as a whole. Biochim Biophys Acta, 2004 May 27, 1663(1-2), 158 - 66 Targeting of immunoliposomes to endothelial cells using a single-chain Fv fragment directed against human endoglin (CD105); Volkel T et al.; We generated immunoliposomes targeting proliferating endothelial cells by chemically coupling a single-chain Fv fragment (scFv A5) directed against human endoglin to the liposomal surface . For this purpose, we introduced an additional cysteine residue at the C-terminus of the scFv fragment . This scFv' fragment was expressed in soluble form in bacteria and allowed for a site-directed coupling to sulfhydryl-reactive lipids incorporated into the lipid bilayer . The immunoliposomes (ILA5) showed rapid and strong binding to human endoglin-expressing endothelial cells (HUVEC, HDMEC), while no binding was observed with various endoglin-negative cell lines and blood lymphocytes . In vitro, ILA5 were stable for several hours in serum- or plasma-containing medium . Incubation of endothelial cells with ILA5 at 37 degrees C led to increased binding and internalisation of the liposomes as evidenced by a perinuclear accumulation . In vitro, doxorubicin-loaded ILA5 showed an increased cytotoxicity towards endothelial cells compared to untargeted liposomes and free doxorubicin . Since the vasculature of tumours is easily accessible to drug carrier systems, the described endothelial cell-specific immunoliposomes may be useful for the development of efficacious and safe vascular targeting agents in cancer therapy. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2004 Apr, 12(2), 185 - 7 {Pulmonary complications in haploidentical bone marrow transplantation}; Wang HX et al.; To explore the occurrence patterns of pulmonary complications at different stages in haploidentical bone marrow transplantation, a series of clinical data as the onset time, etiology, management choices and prognosis in 18 patients with pulmonary disorders were summarized . The results showed that in 18 out of 70 patients after bone marrow transplantation occurred pulmonary complications which included pneumonia affected by bacteria (7 cases), fungus (5 cases) and cytomegalovirus (CMV, 4 cases), bronchiolitis obliterans organizing pneumonia (BOOP, 1 case), and idiopathic pneumonia syndrome (1 case), out of which 8 cases died . Fungal and CMV pneumonia occurred predominantly 2 to 3 months after transplantation, whereas bacterial pneumonia was observed in the duration of 3 to 12 months and 4 cases of them suffered from secondary fungal infections during treatment . BOOP and idiopathic pneumonia syndrome were diagnosed 12 months and 50 days after transplantation respectively . In conclusion: pulmonary complications were commonly seen in haploidentcal bone marrow transplantation, and fungal pneumonia might be the main cause that needs intensive management. Biochemistry, 2004 Jun 1, 43(21), 6511 - 8 The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability; Adinolfi S et al.; Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding . Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans . Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease . The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps . Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities . Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange . By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins . Thermal stability is also gained by the addition of Fe(2+) . This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations . We discuss the implications that our findings have for the role of frataxins as iron-binding proteins. Helicobacter, 2004 Feb, 9(1), 81 - 6 Serological responses of FldA and small-molecular-weight proteins of Helicobacter pylori: correlation with the presence of the gastric MALT tissue; Yang HB et al.; PURPOSE: We tested whether the serological response to Flavodoxin-A (FldA) protein and anti-Helicobacter pylori immunoblots correlated to the degree of mucosaassociated lymphoid tissue (MALT) in the stomach . METHODS: Eighty H . pyloni-infected patients with different degrees of MALT in the stomach were investigated with serum sampling and endoscopy on enrolment, the 2nd and the 12th months after anti-H . pylori therapy . All sera were tested for the anti-FldA protein and anti-H . pylon immunoblots, including 19.5, 26.5, 30, 35, 89, and 116 KDa (CagA) . Regression of follicular gastritis was assessed by histology . RESULTS: Patients with dense lymphoid follicles had higher prevalence rates of anti-FldA protein, 19.5, 26.5, and 30 KDa antibodies of H . pylori (p < .05) . Histologic downgrade of MALT was observed in 25% (10/40) of patients in the 2nd month and in 60% (23/38) in the 12th month after H . pylori therapy . After H . pylori eradication, the patients with MALT and dense lymphoid follicles had significantly negative seroconversions of 19.5, 26.5, 30, and 35 KDa antibodies (p < .05), but not of CagA and FldA . CONCLUSION: Patients with gastric MALT and dense lymphoid follicles had different anti-H . pylori serological responses to those with scanty or an absence of lymphoid follicles . The negative seroconversion of the smaller-molecular-weight proteins, but not CagA and FldA, may correlate with the regression of MALT by H . pylori eradication. Methods Mol Biol, 2004, 268, 189 - 97 Immunomagnetic separation of pathogenic organisms from environmental matrices; Yakub GP et al.; One of the most difficult challenges in the analysis of environmental samples is to separate the organism of interest from a sample that is high in background debris . Immunomagnetic separation (IMS) is one technique that has been developed to accomplish this in a rapid and reliable assay.Immunomagnetic separation (or biomagnetic separation) involves a superparamag-netic, monodispersed, polystyrene microsphere that is coated with a specific ligand . When added to a heterogeneous target suspension, the microspheres bind to the desired target . Using a powerful magnet, the microsphere-target complex is then removed from the suspension . Many different targets of interest can be isolated with this technique, including fungal/bacterial cells or spores, protozoan parasites, cellular and subcellular material, proteins, and nucleic acid products . This wide range of application makes IMS one of the most versatile techniques available for the purification of target products from heterogeneous sample matrices. Methods Mol Biol, 2004, 268, 141 - 51 Intracellular multiplication of Legionella species and the influence of amoebae on their intracellular growth in human monocytes: mono mac 6 cells and Acanthamoeba castellanii as suitable in vitro models; Neumeister B; Legionellae are important etiological agents of pneumonia . Legionella pneumophila (predominantly serogroup 1) is detected in most cases of legionellosis; other species only occasionally cause infections, predominantly in immunocompromized patients . Aquiferous technical systems are the primary source of infection (air-conditioning systems, refrigerators, showers, whirlpools, springs, taps, moisturizing equipment, medical nebulizers, and swimming pools) . Legionellae are present in the water in these systems, within the amoebae, flagellates, and ciliates in which they replicate . After inhalation of contaminated aerosols, the bacteria multiply intracellularly within alveolar macrophages . The ability to multiply within monocytic host cells is usually considered to correspond to pathogenicity . The mechanisms of intracellular replication have been only partially characterized.Analysis of the molecular pathogenesis of Legionella infection, both in the pathogen itself and in the host cell, is the subject of current research and may lead to new options in prophylaxis and treatment . We have established the human Mono Mac 6 cell line (MM6) instead of the previously used histiocytic lymphoma cell line U 937 or the promyelocytic leukemia cell line HL-60 to investigate the intracellular replication of legionellae and the molecular pathogenesis of Legionella infection within human monocytic host cells . MM6 cells represent a more mature macrophage-like cell line that expresses phenotypic and functional properties of mature monocytes and that does not need to be stimulated by phorbol esters or 1,25-dihydroxyvitamin D3 . A good correlation between the prevalence of a given Legionella species and its intracellular multiplication in MM6 cells could be demonstrated.In addition to Legionella, MM6 cells were found to support the intracellular growth of Mycobacterium tuberculosis and Chlamydia pneumoniae, two other important bacterial agents involved in induction of pneumonia . Therefore, the MM6 model might be adaptable to investigations of the molecular pathogenesis of other intracellular bacteria that can replicate within human monocytes and induce disease. Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8281 - 4 Epub 2004 May 20. Magnetic tests for magnetosome chains in Martian meteorite ALH84001; Weiss BP et al.; Transmission electron microscopy studies have been used to argue that magnetite crystals in carbonate from Martian meteorite ALH84001 have a composition and morphology indistinguishable from that of magnetotactic bacteria . It has even been claimed from scanning electron microscopy imaging that some ALH84001 magnetite crystals are aligned in chains . Alignment of magnetosomes in chains is perhaps the most distinctive of the six crystallographic properties thought to be collectively unique to magnetofossils . Here we use three rock magnetic techniques, low-temperature cycling, the Moskowitz test, and ferromagnetic resonance, to sense the bulk composition and crystallography of millions of ALH84001 magnetite crystals . The magnetic data demonstrate that although the magnetite is unusually pure and fine-grained in a manner similar to terrestrial magnetofossils, most or all of the crystals are not arranged in chains. Mol Pharmacol, 2004 Jun, 65(6), 1336 - 43 Discovery of novel selective inhibitors of human intestinal carboxylesterase for the amelioration of irinotecan-induced diarrhea: synthesis, quantitative structure-activity relationship analysis, and biological activity; Wadkins RM et al.; The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-{4-(1-piperidino)-1-piperidino}carbonyloxy-camptothecin (irinotecan; CPT-11) is delayed diarrhea . This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine . After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity . Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent . In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE) . Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase . Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental Ki values (r2 = 0.944) for hiCE . Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses . These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications. Infect Immun, 2004 Jun, 72(6), 3524 - 30 Effect of antibody on the rickettsia-host cell interaction; Feng HM et al.; A recent study demonstrated that polyclonal antibodies to Rickettsia conorii and monoclonal antibodies to outer membrane proteins A (OmpA) and B (OmpB) provided effective, Fc-dependent, passive immunity, even in severe combined immunodeficient mice with an established infection . In order to determine the mechanism of protection, mouse endothelial and macrophage-like cell lines were infected with R . conorii that had been exposed to polyclonal antibodies, monoclonal antibodies to OmpA or OmpB, Fab fragments of the polyclonal antibodies, or normal serum or that were left untreated . At 0 h, Fc-dependent antibody enhancement of R . conorii adherence to endothelial and macrophage-like cell lines was inhibited by the presence of normal serum, suggesting Fc receptor-mediated adherence of opsonized rickettsiae . At 3 h, the opsonized rickettsiae had been internalized . After 72 h, inhibited survival of rickettsiae exposed to polyclonal antibodies or monoclonal antibodies to OmpA or OmpB was evident compared with growth of untreated and normal serum-treated and polyclonal Fab antibody-treated R . conorii . Polyclonal antibodies and an anti-OmpB monoclonal antibody inhibited the escape of R . conorii from the phagosome, resulting in intraphagolysosomal rickettsial death . At 48 h of infection, rickettsicidal activity of macrophages by opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, mannitol, or supplemental L-tryptophan, and endothelial rickettsicidal activity against opsonized rickettsiae was inhibited by NG-monomethyl-L-arginine, superoxide dismutase, catalase, or supplemental L-tryptophan . Thus, Fc-dependent antibodies protected against R . conorii infection of endothelium and macrophages by opsonization that inhibited phagosomal escape and resulted in phagolysosomal killing mediated by nitric oxide, reactive oxygen intermediates, and L-tryptophan starvation. Infect Immun, 2004 Jun, 72(6), 3204 - 17 Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages; Clemens DL et al.; Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages . We have examined the interaction of a recent virulent clinical isolate of F . tularensis subsp . tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy . We assessed the maturation of the F . tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D . Two to four hours after infection, vacuoles containing live F . tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D . However, after 4 h, the colocalization of LAMPs with live F . tularensis organisms declined dramatically . In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h) . We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents . Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F . tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7) . An ultrastructural analysis of the F . tularensis compartment revealed that during the first 4 h after uptake, the majority of F . tularensis bacteria reside within phagosomes with identifiable membranes . The cytoplasmic side of the membranes of approximately 50% of these phagosomes was coated with densely staining fibrils of approximately 30 nm in length . In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment . By 8 h after infection, the majority of live F . tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm . These results indicate that F . tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm . The capacity of F . tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development. Infect Immun, 2004 Jun, 72(6), 3138 - 46 Borrelia burgdorferi binds to, invades, and colonizes native type I collagen lattices; Zambrano MC et al.; Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans . We investigated the interactions between B . burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices . B . burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans . Moreover, following adhesion to these matrices, B . burgdorferi grew and formed microcolonies . The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B . burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices . B . burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans . Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions . Proteinase K treatment of B . burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B . burgdorferi to interact with intact collagen matrices . The high efficiency of binding of B . burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions. J Am Vet Med Assoc, 2004 May 15, 224(10), 1644 - 50, 1606 Cryptobia iubilans infection in juvenile discus; Yanong RP et al.; Four commercial producers of discus (Symphysodon aequifasciatus) were found to have fish infested with the flagellate Cryptobia iubilans . Affected fish had granulomatous gastritis, and many also had granulomatous disease of other organs . The parasite had to be differentiated from the related flagellates Spironucleus spp, which induce different lesions . Transmission electron microscopy was found to be useful in detecting and identifying the parasite . Morbidity and mortality rates in the various fish populations appeared to be linked to a number of variables, including water quality, presence of other parasites and bacteria, diet, species, size, and age of the fish, and optimization of husbandry appeared to be important in alleviating the severity of disease . Metronidazole was not effective for treatment of C iubilans, but bath treatments with dimetridazole (80 mg/L for 24 hours, repeated daily for 3 days) or 2-amino-5-nitrothiazol (10 mg/L for 24 hours, repeated daily for 3 days) may be useful in decreasing the prevalence of infestation. J Am Vet Med Assoc, 2004 May 15, 224(10), 1615 - 22 Gallbladder mucocele in dogs: 30 cases (2000-2002); Pike FS et al.; OBJECTIVE: To determine long-term outcome of dogs with gallbladder mucocele . DESIGN: Retrospective study . ANIMALS: 30 dogs with gallbladder mucocele, including 23 that underwent cholecystectomy . PROCEDURE: Medical records were reviewed for signalment, history, and clinical, ultrasonographic, and surgical findings . Follow-up information was obtained for all dogs that survived the perioperative hospitalization period . RESULTS: 23 dogs had signs of systemic illness; 7 had no clinical signs . Median values for serum activities of alanine aminotransferase and alkaline phosphatase, serum total bilirubin concentration, and total WBC count were significantly higher among dogs with gallbladder rupture than among dogs without rupture . Sensitivity of sonography for detection of rupture was 85.7% . Overall perioperative mortality rate for dogs that underwent cholecystectomy was 21.7%; mortality rate was not significantly greater for dogs with rupture . Aerobic bacteria were isolated from the bile or gallbladder wall in 8.7% of dogs . All 18 dogs discharged from the hospital had complete resolution of clinical signs . In dogs that underwent in-hospital reexamination, serum liver enzyme activities were significantly decreased, compared with preoperative activities . Persistent increases in serum activities of 1 or more liver enzymes were detected in 9 of 12 dogs; 6 of 12 dogs had persistent abnormalities in hepatic echogenicity . Mean follow-up period was 13.9 months . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cholecystectomy is an effective treatment for gallbladder mucocele . Although perioperative mortality rate is high, prognosis after discharge from the hospital is excellent . Rupture of the gallbladder warrants emergency surgical intervention but does not preclude a positive outcome. Evolution Int J Org Evolution, 2004 Apr, 58(4), 790 - 8 Haplodiploidy as an outcome of coevolution between male-killing cytoplasmic elements and their hosts; Normark BB; Haplodiploidy (encompassing both arrhenotoky and paternal genome elimination) could have originated from coevolution between male-killing endosymbiotic bacteria and their hosts . In insects, haplodiploidy tends to arise in lineages that rely on maternally transmitted bacteria for nutrition and that have gregarious broods in which competition between siblings may occur . When siblings compete, there is strong selection on maternally transmitted elements to kill males . I consider a hypothetical bacterial phenotype that renders male zygotes effectively haploid by preventing chromosome decondensation in male-determining sperm nuclei . By causing high male mortality, such a phenotype can be advantageous to the bacterial lineage . By eliminating paternal genes, it can also be advantageous to the host female . A simple model shows that the host female will benefit under a wide range of values for the efficiency of resource re-allocation, the efficiency of transmission, and the viability of haploid males . This hypothesis helps to explain the ecological correlates of the origins of haplodiploidy, as well as such otherwise puzzling phenomena as obligate cannibalism by male Micromalthus beetles, reversion to diploidy by aposymbiotic male stictococcid scale insects, and the bizarre genomic constitution of scale insect bacteriomes. J Immunol, 2004 Jun 1, 172(11), 7069 - 77 Iron chelator triggers inflammatory signals in human intestinal epithelial cells: involvement of p38 and extracellular signal-regulated kinase signaling pathways; Choi EY et al.; Competition for cellular iron (Fe) is a vital component of the interaction between host and pathogen . Most bacteria have an obligate requirement for Fe to sustain infection, growth, and survival in host . To obtain iron required for growth, many bacteria secrete iron chelators (siderophores) . This study was undertaken to test whether a bacterial siderophore, deferoxamine (DFO), could trigger inflammatory signals in human intestinal epithelial cells as a single stimulus . Incubation of human intestinal epithelial HT-29 cells with DFO increased the expression of IL-8 mRNA, as well as the release of IL-8 protein . The signal transduction study revealed that both p38 and extracellular signal-regulated kinase-1/2 were significantly activated in response to DFO . Accordingly, the selective inhibitors for both kinases, either alone or in combination, completely abolished DFO-induced IL-8 secretion, indicating an importance of mitogen-activated protein kinases pathway . These proinflammatory effects of DFO were, in large part, mediated by activation of Na(+)/H(+) exchangers, because selective blockade of Na(+)/H(+) exchangers prevented the DFO-induced IL-8 production . Interestingly, however, DFO neither induced NF-kappaB activation by itself nor affected IL-1beta- or TNF-alpha-mediated NF-kappaB activation, suggesting a NF-kappaB-independent mechanism in DFO-induced IL-8 production . Global gene expression profiling revealed that DFO significantly up-regulates inflammation-related genes including proinflammatory genes, and that many of those genes are down-modulated by the selective mitogen-activated protein kinase inhibitors . Collectively, these results demonstrate that, in addition to bacterial products or cell wall components, direct chelation of host Fe by infected bacteria may also contribute to the evocation of host inflammatory responses. J Immunol, 2004 Jun 1, 172(11), 6838 - 45 Oligosaccharide side chains on human secretory IgA serve as receptors for ricin; Mantis NJ et al.; Secretory IgA (sIgA) Abs are polymeric Igs comprised of two or more IgA monomers joined together at their C termini and covalently associated with a 70-kDa glycoprotein called secretory component . As the predominant Ig type in gastrointestinal sections, sIgA Abs are centrally important in adaptive immunity to enteropathogenic bacteria, viruses, and toxins . In this study, we demonstrate that sIgA Abs may also function in innate defense against ricin, a naturally occurring, galactose-specific plant lectin with extremely potent shiga toxin-like enzymatic activity . In lectin blot overlay assays, we found that ricin bound to secretory component and the H chain of human IgA, and this binding was inhibited by the addition of excess galactose . The toxin also recognized IgM (albeit with less affinity than to IgA), but not IgG . Ricin bound to both human IgA1 and IgA2, primarily via N-linked oligosaccharide side chains . At 100-fold molar excess concentration, sIgA (but not IgG) Abs inhibited ricin attachment to the apical surfaces of polarized intestinal epithelial cells grown in culture . sIgA Abs also visibly reduced toxin binding to the luminal surfaces of human duodenum in tissue section overlay assays . We conclude that sIgA Abs in mucosal secretions may serve as receptor analogues for ricin, thereby reducing the effective dose of toxin capable of gaining access to glycolipid and glycoprotein receptors on epithelial cell surfaces. Physiol Plant, 2004 Jun, 121(2), 239 - 249 Reduction of dark chilling stress in N-fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics; Van Heerden PD et al.; Sub-optimal night temperatures below 15 degrees C (dark chilling) frequently reduce soybean {Glycine max (L.) Merrill} production . Nitrate application is known to alleviate some of the negative effects of low root zone temperatures, probably by counteracting the inhibition caused by decreased symbiotic nitrogen fixation (SNF) . Under field conditions, however, dark chilling is frequently not accompanied by low root zone temperatures . The possibility that nitrate might increase dark-chilling tolerance under these conditions is still largely unexplored . In addition to quantifying vegetative development by means of the plastochron index, O-J-I-P (O-I(1)-I(2)-P) chlorophyll a fluorescence transients were recorded in soybean genotypes of contrasting chilling tolerance during and following exposure to dark chilling in the absence of low root zone temperatures . Plants, inoculated with the N(2)-fixing bacteria, Bradyrhizobium japonicum, were grown with and without nitrate supplementation . The recorded O-J-I-P chlorophyll a fluorescence transients were analysed by the so-called JIP-test which translates stress-induced alterations in these transients to changes in biophysical parameters that quantifies the energy flow through photosystem II (PSII) . One of these parameters, the performance index (PI(ABS)), combines the three main functional steps (light energy absorption, excitation energy trapping, and conversion of excitation energy to electron transport) of photosynthetic activity by a PSII reaction centre complex into a single multiparametric expression . By using the PI(ABS) we could convincingly show that nitrate supplementation considerably enhances dark-chilling tolerance and recovery capacity of plants in the absence of low root zone temperatures . This was especially true for the chilling-sensitive genotype ('Java 29'), suggesting that the response of SNF to dark chilling might be an important factor contributing towards genotypic differences in chilling tolerance . Our results corroborated previous reports about the superior chilling tolerance of 'Maple Arrow', a chilling-tolerant genotype . The results obtained indicated that the PI(ABS) is a far more sensitive indicator of dark-chilling stress than the maximum quantum yield of primary photochemistry (F(V)/F(M)). J Periodontol, 2004 Apr, 75(4), 578 - 85 The in vivo expression of membrane-bound CD14 in periodontal health and disease; Jin L et al.; BACKGROUND: Membrane-bound CD14 (mCD14) is a myeloid differentiation antigen expressed on monocytes/macrophages and neutrophils . It is a key molecule responsible for the innate recognition of bacteria by host cells and functions as an important receptor for bacterial lipopolysaccharide . This study investigated the in vivo expression profile and levels of mCD14 in healthy and diseased gingival tissues . METHODS: Gingival biopsies were obtained from 24 patients with chronic periodontitis, including 22 periodontal pocket tissues, 13 clinically healthy tissues, and 18 inflamed connective tissues (i.e., granulation tissues) . Gingival biopsies from seven periodontally healthy subjects were used as controls . mCD14 was detected by immunohistochemistry . RESULTS: mCD14 was detected in 21 of 22 periodontal pocket tissues and all other categories of tissues . The mCD14-positive cells were mainly confined to the gingival epithelium-connective tissue interface . The expression levels in periodontally healthy subjects were significantly higher than in the patients . Within the patients, clinically healthy tissues showed greater levels of mCD14 than periodontal pocket tissues and granulation tissues . CONCLUSIONS: mCD14 was commonly expressed in both healthy and diseased gingival tissues and was predominantly confined to the epithelium-connective tissue interface . The positive relationship observed between mCD14 expression levels and periodontal health may imply that mCD14 is associated with favorable host responses to bacterial challenge and contributes to maintaining periodontal homeostasis. Proc Natl Acad Sci U S A, 2004 Jun 8, 101(23), 8563 - 8 Epub 2004 May 18. Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD(+)-dependent Sir2 histone/protein deacetylases; Zhao K et al.; Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology . These enzymes bind NAD(+) and acetyllysine within protein targets and generate lysine, 2'-O-acetyl-ADP-ribose, and nicotinamide products . To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD(+) analogue, carba-NAD(+), as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose . The ternary complex with carba-NAD(+) reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues . Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide . A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2'-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved beta1-alpha2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD(+) reactions of nicotinamide cleavage and ADP-ribose transfer to acetate . Together, these studies provide insights into the chemistry of NAD(+) cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules. J Bacteriol, 2004 Jun, 186(11), 3384 - 91 Stress response gene regulation in Chlamydia is dependent on HrcA-CIRCE interactions; Wilson AC et al.; HrcA is a transcriptional repressor that regulates stress response genes in many bacteria by binding to the CIRCE operator . We have previously shown that HrcA regulates the promoter for the dnaK heat shock operon in Chlamydia . Here we demonstrate that HrcA represses a second heat shock promoter that controls the expression of groES and groEL, two other major chlamydial heat shock genes . The CIRCE element of C . trachomatis groEL is the most divergent of known bacterial CIRCE elements, and HrcA had a decreased ability to bind to this nonconsensus operator and repress transcription . We demonstrate that the CIRCE element is necessary and sufficient for transcriptional regulation by chlamydial HrcA and that the inverted repeats of CIRCE are the binding sites for HrcA . Addition of a CIRCE element upstream of a non-heat-shock promoter allowed this promoter to be repressed by HrcA, showing in principle that a chlamydial promoter can be genetically modified to be inducible . These results demonstrate that HrcA is the regulator of the major chlamydial heat shock operons, and we infer that the mechanism of the heat shock response in Chlamydia is derepression . However, derepression is likely to involve more than a direct effect of increased temperature as we found that HrcA binding to CIRCE and HrcA-mediated repression were not altered at temperatures that induce the heat shock response. J Antimicrob Chemother, 2004 Jun, 53 Suppl 2, ii75 - 81 Safety and tolerability of ertapenem; Teppler H et al.; Ertapenem is a Group 1 carbapenem that was licensed in the USA in November 2001 and in Europe in April 2002 . Its safety profile has been assessed in 240 healthy volunteers participating in 12 clinical pharmacology studies and in 2046 patients enrolled in five Phase IIa and eight Phase IIb/III clinical trials . The most common drug-related adverse events (AEs) reported in trials comparing ertapenem and piperacillin-tazobactam and in trials comparing ertapenem and ceftriaxone were: diarrhoea (ertapenem versus piperacillin-tazobactam 5.0% versus 7.0%; ertapenem versus ceftriaxone 5.6% versus 5.9%); infused vein complications (ertapenem versus piperacillin-tazobactam 4.5% versus 7.9%; ertapenem versus ceftriaxone 3.2% versus 4.6%); nausea (ertapenem versus piperacillin-tazobactam 2.5% versus 3.4%; ertapenem versus ceftriaxone 3.4% versus 3.3%); and elevations in alanine aminotransferase levels (ertapenem versus piperacillin-tazobactam 8.8% versus 7.3%; ertapenem versus ceftriaxone 8.3% versus 6.9%) . Most ertapenem-related AEs were reported as mild-to-moderate in intensity . Ertapenem was not associated with prolongation of the QTc interval . Local reactions of moderate-to-severe intensity at the infusion site were infrequent and occurred with similar frequency in the ertapenem and comparator treatment groups . No overall differences in safety were observed between elderly (aged > or = 65 years and > or = 75 years) and younger patients . Ertapenem, 1 g once a day given by intravenous infusion or intramuscular injection, was generally well tolerated and had overall safety and tolerability profiles similar to those of piperacillin-tazobactam and ceftriaxone. J Evol Biol, 2004 May, 17(3), 692 - 700 Wolbachia affects oviposition and mating behaviour of its spider mite host; Vala F et al.; Wolbachia bacteria are transmitted from mother to offspring via the cytoplasm of the egg . When mated to males infected with Wolbachia bacteria, uninfected females produce unviable offspring, a phenomenon called cytoplasmic incompatibility (CI) . Current theory predicts that 'sterilization' of uninfected females by infected males confers a fitness advantage to Wolbachia in infected females . When the infection is above a threshold frequency in a panmictic population, CI reduces the fitness of uninfected females below that of infected females and, consequently, the proportion of infected hosts increases . CI is a mechanism that benefits the bacteria but, apparently, not the host . The host could benefit from avoiding incompatible mates . Parasite load and disease resistance are known to be involved in mate choice . Can Wolbachia also be implicated in reproductive behaviour? We used the two-spotted spider mite - Wolbachia symbiosis to address this question . Our results suggest that uninfected females preferably mate to uninfected males while infected females aggregate their offspring, thereby promoting sib mating . Our data agrees with other results that hosts of Wolbachia do not necessarily behave as innocent bystanders - host mechanisms that avoid CI can evolve. Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7913 - 8 Epub 2004 May 17. Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone; Magnusson OT et al.; The biosynthesis of pyrroloquinoline quinone (PQQ), a vitamin and redox cofactor of quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes, pqqA to -F . PqqC, the protein encoded by pqqC, catalyzes the final step in the pathway in a reaction that involves ring cyclization and eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic-acid to PQQ . Herein, we describe the crystal structures of PqqC and its complex with PQQ and determine the stoichiometry of H2O2 formation and O2 uptake during the reaction . The PqqC structure(s) reveals a compact seven-helix bundle that provides the scaffold for a positively charged active site cavity . Product binding induces a large conformational change, which results in the active site recruitment of amino acid side chains proposed to play key roles in the catalytic mechanism . PqqC is unusual in that it transfers redox equivalents to molecular oxygen without the assistance of a redox active metal or cofactor . The structure of the enzyme-product complex shows additional electron density next to R179 and C5 of PQQ, which can be modeled as O2 or H2O2, indicating a site for oxygen binding . We propose a reaction sequence that involves base-catalyzed cyclization and a series of quinone-quinol tautomerizations that are followed by cycles of O2/H2O2-mediated oxidations. Am J Surg, 2004 May, 187(5A), 65S - 70S Understanding chronic wounds: a unifying hypothesis on their pathogenesis and implications for therapy; Mustoe T; The development of new therapies for treatment of chronic wounds has not matched the availability of treatment modalities forecast by the pharmaceutical industry . This is attributable in large part to difficulties encountered in clinical trials as well as in isolating study design variables . Our hypothesis attempts to address this shortcoming . We are proposing that chronic wound pathogenesis is based on 3 fundamental factors: the cellular and systemic changes of aging, repeated ischemia-reperfusion injury, and bacterial colonization with resulting inflammatory host response . The derivation of this hypothesis is founded on the observation that the 3 primary categories of chronic wounds--pressure ulcers, diabetic ulcers, and venous ulcers, which are the overwhelming majority of chronic wounds--have these common causative factors . Our hypothesis incorporates major implications for treatment modalities based on these factors . Addressing the first issue, the cellular and systemic changes of aging, Regranex (Ortho-McNeil Pharmaceutical, Inc, Raritan, NJ), a platelet-derived growth factor drug, has shown great promise . Additional treatment modalities that address the second and third problems, repeated ischemia-reperfusion injury and bacterial colonization, include vacuum-assisted closure, warming of local tissue, and water irrigation using pulsed lavage . Additionally, treatment comprising a combination of these approaches has demonstrated success. Spectrochim Acta A Mol Biomol Spectrosc, 2004 Jun, 60(7), 1563 - 71 EPR and electronic spectral studies on Co(II), Ni(II) and Cu(II) complexes with a new tetradentate {N4} macrocyclic ligand and their biological activity; Chandra S et al.; Cobalt(II), nickel(II) and copper(II) complexes having the general composition M(L)X2 (where M = CO(II), Ni(II) and Cu(II), L = ligand, i.e . 3,4,12,13-tetraketo-2,5,11,14,19,20-hexaazatricyclo{13.3.1.1(6-10)}cosane; 1(19),6,8,10(20),15,17-hexaene and X stands for Cl-; NO3- and SO42-), have been prepared . The structure of the complexes has been elucidated by elemental analysis, molar conductance, magnetic susceptibility measurements, mass, IR, electronic and EPR spectral studies . The magnetic moment measurements of the complexes indicate that the metal ion is in high-spin state . On the basis of IR, electronic and EPR spectral studies an octahedral geometry was assigned for Co(II) and Ni(II) complexes whereas tetragonal geometry for Cu(II) complexes . This ligand and its complexes were also screened against bacteria and pathogenic fungi in vitro. Clin Exp Immunol, 2004 Jun, 136(3), 472 - 82 Recognition of nonpeptide antigens by human V gamma 9V delta 2 T cells requires contact with cells of human origin; Green AE et al.; SUMMARY It is becoming apparent that gamma delta T cells form an important part of the adaptive immune response . However, the ligands recognized by gamma delta T cell receptors (TCRs) and the exact biological function of the cells that express this receptor remain unclear . Numerous studies have shown that the dominant human peripheral blood subset of gamma delta T cells, which express a V gamma 9V delta 2 TCR, can activate in response to low molecular weight nonpeptidic molecules . Some of these components have been purified from bacteria or parasites . We examined the activation of polyclonal gamma delta T cell lines, clones with V gamma 9V delta 2 and V gamma 9V delta 1 TCRs, and gamma delta T cells directly ex vivo in response to multiple phosphate, alkylamine and aminobisphosphonate (nBP) antigens and purified protein derivative from Mycobacterium tuberculosis (PPD) . V gamma 9V delta 2 T cells were able to respond to multiple small organic molecules of highly variable structure whereas cells expressing a similar V gamma 9 chain paired with a V delta 1 chain failed to recognize these antigens . Thus, the TCR delta chain appears to make an important contribution to the recognition of these antigens . The kinetics of responses to alkylphosphate and alkylamine antigens differ from those of responses to the nBP pamidronate . These different classes of antigen are believed to have differed mechanisms of action . Such differences explain why nBPs can be pulsed onto antigen presenting cells (APCs) and still retain their ability to activate gamma delta T cells while alkylphosphate and alkylamine antigens cannot . We also demonstrate that a substantial proportion of the cells that produce IFN gamma directly ex vivo in response to PPD are gamma delta T cells and that gamma delta T cell activation requires contact with cells of human origin. Equine Vet J, 2004 Apr, 36(3), 267 - 72 The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse; Bailey SR et al.; REASONS FOR PERFORMING STUDY: Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis . However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined . HYPOTHESIS: Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo . METHODS: Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v . into standing nonsedated horses . Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored . Plasma 5-hydroxytryptamine (5-HT) concentrations were measured by HPLC . RESULTS: Tryptamine and phenylethylamine infusions had no effect on systemic arterial blood pressure or heart rate, but caused significant decreases in digital arterial blood flow (mean +/- s.e . 29.2 +/- 8.5 and 18.4 +/- 6.8%, respectively) . Both amines also caused decreases in dorsal hoof wall temperature (0.6 +/- 0.1 and 0.5 +/- 0.1 degrees C for tryptamine and phenylethylamine, respectively) and concomitant increases in plasma 5-HT concentration . CONCLUSIONS: Tryptamine and phenylethylamine caused reduction of digital blood flow, effects which may have been mediated, in part, via displacement of 5-HT from platelets . POTENTIAL RELEVANCE: Amine compounds occurring in the equine hindgut, if released into the circulation following carbohydrate overload, could contribute to selective digital vasoconstriction . Further work in ponies and horses, with naturally occurring laminitis, is necessary to determine whether amines represent a therapeutic target in this disease. Int J Med Microbiol, 2004 Apr, 293 Suppl 37, 120 - 5 Increased expression of Borrelia burgdorferi factor H-binding surface proteins during transmission from ticks to mice; Miller JC et al.; The spirochete Borrelia burgdorferi is transmitted to humans and other warm blooded animals through the bites of infected Ixodes species ticks . Our studies indicate that these spirochetes utilize a quorum sensing mechanism to control protein expression patterns that involves the chemical signal autoinducer-2 (AI-2) . Through this mechanism, a population of Lyme disease spirochetes may synchronize production of proteins needed for infection processes . AI-2 is produced by the B . burgdorferi LuxS protein, which we have demonstrated to be a functional enzyme . It has also been previously reported that luxS message is upregulated in feeding nymphal ixodid ticks . Among the B . burgdorferi proteins regulated through AI-2 are the complement inhibitory factor H binding Erp lipoproteins . We now report Erp protein expression is also increased during transmission of B . burgdorferi from nymphal ticks to mammalian hosts . Essentially no B . burgdorferi within unfed nymphal ticks expressed Erps, while almost all transmitted bacteria were Erp positive . These studies suggest that B . burgdorferi within feeding nymphal ticks produce AI-2 to coordinate expression of mammalian infection associated proteins, such as the factor H binding Erp lipoproteins . Binding of mammalian host factor H by Erps may then help promote bacterial dissemination through host tissues. Int J Med Microbiol, 2004 Apr, 293 Suppl 37, 86 - 92 Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp . in Southern Germany; Hartelt K et al.; Tick-transmitted diseases like tick-borne encephalitis and Lyme borreliosis have been well known in Germany for decades . Ongoing research now gives an additional focus to a broad range of other bacteria and parasites in ticks like Anaplasma phagocytophilum, former Ehrlichia sp., Rickettsia sp . and Babesia sp . Knowledge about the prevalence of these infectious agents in ticks is an important prerequisite for risk assessment of human diseases . Therefore nymphs and adult Ixodes ricinus ticks were collected and examined for Anaplasma phagocytophilum (n = 5424 ticks), Rickettsia sp . (n = 1187), and Babesia sp . (n = 3113) . For the detection of Anaplasma phagocytophilum, DNA from the 16S rDNA gene was amplified by nested PCR and hybridized with a DIG-labeled oligonucleotide probe . The examination of Rickettsia sp . was performed by single PCR . A partial sequence of the citrate synthase gene was amplified . As a target for the detection of Babesia sp., DNA from the 18S rDNA gene was amplified, also by single PCR . All positive PCR products were sequenced to control specificity . Anaplasma phagocytophilum was detected by PCR in n = 103 (1.9%) out of 5,424 examined ticks from 11 investigation areas . However, not all positive PCR products hybridized using DIG-labeled oligonucleotide probe . Thus, the result of sequencing indicated that only 1.0% (n = 54) belonged to Anaplasma phagocytophilum and nearly half of these PCR products (0.9%) were identified as Wolbachia sp . Rickettsia sp . in Ixodes ricinus ticks from 3 areas were found in n = 105 (8.9%) out of 1,187 ticks examined (range from 13.3% to 5.6%) . Sequencing showed Rickettsia helvetica exclusively . In about 2.6% of Rickettsia-positive ticks, double infection with Anaplasma phagocytophilum was found . Babesia sp . was detected in n= 31 (1.0%) out of 3,113 ticks examined, which originated from 4 different areas . By sequencing, n = 28 (90.0%) were identified as Babesia divergens . Three of all Babesia-positive ticks were identified as harboring Babesia microti . The detection of Anaplasma phagocytophilum, Rickettsia sp . and Babesia sp . demonstrates their possible role as a source of human infection in Germany. Ying Yong Sheng Tai Xue Bao, 2004 Feb, 15(2), 313 - 5 {Suspending particulates in rotifer-culturing pond ecosystem}; Zhao W et al.; A comparative study on the structure and dynamics of suspending particulates in static and current water rotifer-culturing ponds showed that the total content of suspending particulates varied from 6.76 to 65.39 mg.L-1, with an average of 11.50 mg.L-1 in static water ponds, among which, organic pariculates accounted for 66.8%, while inorganic particulartes occupied 33.2% . In the organic particulates, both particulate detritus and bacteria accounted for 31%, while phytoplankton and zooplankton occupied 30.5% and 5.3%, respectively . The amount of suspending particulates in current water ponds was 41.83 mg.L-1 in average, among which, organic pariculates accounted for 70.4%, while inorganic pariculates occupied 29.6% . In the organic pariculates, both particulate detritus and bacteria accounted for 39.2%, while phytoplankton and zooplankton occupied 30% and 1.2%, respectively . The content of suspending pariculates in static water ponds was less than those in current water ponds . Rotifer-culturing pond was a special type of ecosystem, where Brachionus plicatilis was the major consumer, its community structure was sample, and the abiotic factors were easy to be changed, which resulted in the dynamics of suspending pariculates content in ponds . The ratio of organic to total pariculates in rotifer-culturing ponds was higher, and the contents of both particulate detritus and bacteria were corresponding to that of plankton. Bull Acad Natl Med, 2003, 187(7), 1261 - 74; discussion 1274-6 {Molecular mechanisms of bile formation and cholestatic diseases}; Poupon R; Biliary function is a vital function of the liver which results from the vectorial transport of endogenous and exogenous substrates through three compartments sequentially: the vascular space, the cellular space and the biliary space . The biliary function is responsible for the homeostasis of lipid metabolism in particular of cholesterol metabolism, the elimination of toxic endo--and xenobiotics such as (bilirubin, lipid bacteria products (endotoxin)) and several inflammatory mediators . Bile elaborated in canaliculi, is modified by cholangiocytes through secretion and absorption . Bile is essential for the intestinal digestion and absorption of nutriments . The main determinant of bile formation is an osmotic filtration process resulting from active transport of bile acids and other osmotic solutes (glutathione) . Most of the membrane transporters ensuring bile formation have now been identified . The expression of these membrane transporters is regulated through transcriptional and post-tranductional mechanisms . Transcriptional regulation is under the control of nuclear receptors activated by ligands such as bile acids, which act as endogenous steroids synthesized from cholesterol in hepatocytes . Cholestatic liver diseases comprise genetic diseases resulting from the complex interaction between genetic and environmental factors . Monogenic cholestatic diseases recently identified illustrate the key role of membrane transporters in biliary function . Bile acids and inflammatory mediators are potent modulators transporters and nuclear receptor genes and thus trigger an adaptative response to cholestasis . The extent of this adaptative response could explain the compelling phenotypic variability of cholestatic diseases in childhood and adults . The firstline medical treatment is currently ursodeoxycholic acid and in case of failure of this medical treatment, liver transplantation is required . Recent progress in the molecular pathogenesis of bile formation and cholestatic liver diseases is expected to provide the design of drugs targeted to the molecular abnormalities typical of cholestatic diseases. Proteins, 2004 Jun 1, 55(4), 1063 - 9 Role of protein in the primary step of the photoreaction of yellow protein; Yamada A et al.; We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination . The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method . The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself . As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term . This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments . We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum . Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment . Proteins, 2004 Jun 1, 55(4), 785 - 91 Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif; Chen S et al.; UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division . There is no structure information available on protein of this family . We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography . Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure . The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer . The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure . Hum Mutat, 2004 Jun, 23(6), 599 - 611 Structural and functional analysis of mutations at the human hypoxanthine phosphoribosyl transferase (HPRT1) locus; Duan J et al.; Hypoxanthine phosphoribosyl transferase (HPRT, also known as HGPRT) is an often-used genetic marker in eukaryotic cells . The gene is conserved from bacteria to human, with retained catalytic activity, although substrate specificity may have changed, and the enzyme is essential in malaria-causing protozoans . Inherited mutations in the human HPRT1 gene result in three different phenotypes: Lesch-Nyhan syndrome (LNS or LND), LND variants, and HPRT-related hyperuricemia (HRH) . In cultured cells, loss of HPRT activity gives rise to 6-thioguanine (6-TG) resistance . In general, cells from LND patients are also 6-TG resistant, whereas cells from HRH patients are not, with some interesting exceptions . Using modeling methods, we have studied the correlation between the mutable and nonmutated amino acid residues on one hand, and sequence conservation and predicted phenotypic effects on the other hand . Our results demonstrate that most of the mutations are explainable by the predicted effect on protein structure and function . They are also consistent with sequence conservation . Moreover, the mutational profiles of TG-resistant cells and LND overlap to a great extent, while most of the mutations in HRH are unique to that condition . We have also noticed a strong correlation between mutations in the tetramer interfaces and observed phenotypes, suggesting a functional role for a tetramer transition during catalysis . J Contam Hydrol, 2004 Jul, 71(1-4), 219 - 37 Impact of edible oil injection on the permeability of aquifer sands; Coulibaly KM et al.; Recent laboratory and field studies have shown that food-grade edible oils can be injected into the subsurface for installation of in-situ permeable reactive barriers . However to be effective, the oil must be distributed out away from the oil injection points without excessive permeability loss . In this work, we examine the distribution of soybean oil in representative aquifer sediments as non-aqueous phase liquid oil (NAPL oil) or as an oil-in-water emulsion . Laboratory columns packed with sands or clayey sands were flushed with either NAPL oil or a soybean emulsion followed by plain water, while monitoring permeability loss and the final oil residual saturation . NAPL oil can be injected into coarse-grained sands . However NAPL injection into finer grained sediments requires high injection pressures which may not be feasible at some sites . In addition, NAPL injection results in high oil residual saturations and moderate permeability losses . In contrast, properly prepared emulsions can be distributed through sands with varying clay content without excessive pressure buildup, low oil retention and very low to moderate permeability loss . For effective transport, the emulsion must be stable, the oil droplets must be significantly smaller than the mean pore size of the sediment and the oil droplets should have a low to moderate tendency to stick to each other and the aquifer sediments . In our work, oil retention and associated permeability loss increased with sediment clay content and with the ratio of droplet size to pore size . For sandy sediments, the permeability loss is modest (0-40% loss) and is proportional to the oil residual saturation . Vet Microbiol, 2004 Jun 3, 100(3-4), 269 - 82 Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR; Oidtmann B et al.; A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci . A set of oligonucleotide primers was designed to specifically amplify A . astaci DNA in the ITS region surrounding the 5.8S rDNA gene . The PCR amplifies a 115bp amplicon . The specificity of the primers was demonstrated by testing on 27 A . astaci strains and against 20 non-A . astaci Oomycetes and 5 fungal species . Most of the non-A . astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment . Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish . A protocol for the extraction of A . astaci DNA from infected crayfish tissue was developed . The optimised method allows the detection of two genome equivalents of purified A . astaci genomic DNA . The method was tested on noble crayfish (Astacus astacus), artificially infected with A . astaci . Detection of A . astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure. Vet Microbiol, 2004 Jun 3, 100(3-4), 197 - 204 Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis; Vansnick E et al.; Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map) . The primers used for IS900 detection of Map have amplified these sequences causing false positive results . In this study, we have developed two new PCR assays for the detection of Map . The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence . The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates) . All Map strains were positive and all non-Map strains were negative . Serial dilutions of Map bacteria were used to assess the sensitivity of the assays . We achieved a sensitivity of 1CFU per PCR for both assays . In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers . The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done. J Colloid Interface Sci, 2004 Jun 15, 274(2), 442 - 50 Reaction of hydroquinone with hematite; II . Calculated electron-transfer rates and comparison to the reductive dissolution rate; Stack AG et al.; The rate of reaction of hematite with quinones and the quinone moieties of larger molecules may be an important factor in limiting the rate of reductive dissolution of hematite, especially by iron-reducing bacteria . It is possible that the rate of reductive dissolution of hematite in the presence of excess hydroquinone at pH 2.5 may be limited by the electron-transfer rate . Here, a reductive dissolution rate was measured and compared to electron-transfer rates calculated using Marcus theory . An experimental rate constant was measured at 9.5 x 10 (-6) s(-1) and the reaction order with respect to the hematite concentration was found to be 1.1 . Both the dissolution rate and the reaction order of hematite concentration compare well with previous measurements . Of the Marcus theory calculations, the inner-sphere part of the reorganization energy and the electronic coupling matrix element for hydroquinone self-exchange electron transfer are calculated using ab initio methods . The second order self-exchange rate constant was calculated to be 1.3 x 10 (7) M(-1)s(-1), which compares well with experimental measurements . Using previously published data calculated for hexaquairon(III)/(II), the calculated electron-transfer rate for the cross reaction with hydroquinone also compares well to experimental measurements . A hypothetical reductive dissolution rate is calculated using the first-order electron-transfer rate constant and the concentration of total adsorbed quinone . Three different models of the hematite surface are used as well as multiple estimates for the reduction potential, the surface charge, and the adsorption density of hydroquinone . No calculated dissolution rate is less than five orders of magnitude faster than the experimentally measured one. J Colloid Interface Sci, 2004 Jun 15, 274(2), 433 - 41 Reaction of hydroquinone with hematite; I . Study of adsorption by electrochemical-scanning tunneling microscopy and X-ray photoelectron spectroscopy; Stack AG et al.; The reaction of hematite with quinones and the quinone moieties of larger molecules may be an important factor in limiting the rate of reductive dissolution, especially by iron-reducing bacteria . Here, the electrochemical and physical properties of hydroquinone adsorbed on hematite surfaces at pH 2.5-3 were investigated with cyclic voltammetry (CV), electrochemical-scanning tunneling microscopy (EC-STM), and X-ray photoelectron spectroscopy (XPS) . An oxidation peak for hydroquinone was observed in the CV experiments, as well as (photo)reduction of iron and decomposition of the solvent . The EC-STM results indicate that hydroquinone sometimes forms an ordered monolayer with approximately 1.1 QH(2)/nm(2), but can be fairly disordered (especially when viewed at larger scales) . XPS results indicate that hydroquinone and benzoquinone are retained at the interface in increasing amounts as the reaction proceeds, but reduced iron is not observed . These results suggest that quinones do not adsorb by an inner-sphere complex where adsorbate-surface interactions determine the adsorbate surface structure, but rather in an outer-sphere complex where interactions among the adsorbate molecules dominate. Vox Sang, 2004 May, 86(4), 239 - 45 Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets; Janetzko K et al.; BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs) . To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function . MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units . Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts . In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h . In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h . Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days . RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7 . Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting . CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage. Hepatogastroenterology, 2004 May-Jun, 51(57), 757 - 61 Stress ulcer prophylaxis in critically ill patients: a randomized controlled trial; Kantorova I et al.; BACKGROUND/AIMS: Critically ill patients especially who require mechanical ventilation or have coagulopathy are at increased risk for stress-related gastrointestinal hemorrhage . There are conflicting data on the efficacy and complication rates of various prophylactic regimens . METHODOLOGY: Our single-center randomized, placebo-controlled study included 287 patients with high risk for stress-related upper gastrointestinal hemorrhage (>48 h mechanical ventilation, coagulopathy) . We compared 3 prophylactic regimens (proton pump inhibitor--omeprazole 40 mg i.v . once daily, n=72; H2 antagonists--famotidine 40 mg twice a day, n=71; and sucralfate 1 g every 6 hours, n=69) with placebo (n=75) in patients with trauma or after major surgery . RESULTS: Of 287 assessable patients, clinically significant stress-related upper gastrointestinal bleeding was observed in 1%, 3%, 4%, and 1% of patients assigned to receive omeprazole, famotidine, sucralfate, and placebo, respectively (p>0.28) . Bleeding developed significantly more often in patients with coagulopathy compared with the others (10% vs . 2%; p=0.006) . The gastric pH (p>0.001) and gastric colonization (p<0.05) was significantly higher in the patients who received pH increasing substances when compared with the other 2 groups . Nosocomial pneumonia occurred in 11% of patients receiving omeprazole, in 10% of famotidine patients, in 9% of sucralfate patients and in 7% of controls (p>0.34) . No statistically significant differences were found for days on ventilator, length of ICU stay, or mortality among all the 4 groups . CONCLUSIONS: We could not show that omeprazole, famotidine, or sucralfate prophylaxis can affect already very low incidence of clinically important stress-related bleeding in high-risk surgical intensive care unit patients . Furthermore, our data suggested that especially gastric pH increasing medication could increase the risk for nosocomial pneumonia . Routine prophylaxis for stress-related bleeding even in high-risk patients seems not to be justified. Mol Cell Biol, 2004 Jun, 24(11), 5050 - 9 Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86; Myung K et al.; Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans . In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species . Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression . Thus, it has been unclear which model system is most relevant for humans . We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability . Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the addi