bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of PET imaging in lymphoma
Authors: Burton Catherine , Ell Peter , Linch David
Published: October 2004

Positron emission tomography using fluorine‐18 (FDG‐PET) is increasingly used in the staging and follow‐up of malignant lymphomas, although its precise role has not yet been determined. This review considers the results reported at the different stages in the disease history and separately considers the major histological subtypes. Attention is given to the situations in which PET scanning is most likely to influence management. Finally, this review discusses ongoing developments in PET scanning with improved resolution and different radiolabelled tracers.
FLT3 mutations in myeloid sarcoma
Authors: Ali Ansari‐Lari M. , Yang Ching‐Fen , Tinawi‐Aljundi Rima , Cooper Lisa , Long Patricia , Allan Robert H. , Borowitz Michael J. , Berg Karin D. , Murphy Kathleen M.
Published: October 2004

Myeloid sarcoma is an extramedullary tumour that typically occurs in the setting of acute myeloid leukaemia (AML), or myeloproliferative disorders. In AML, two types of mutations in Fms‐like tyrosine kinase 3 (FLT3) have been described; internal tandem duplications (ITD) and point mutations at aspartic acid residue 835 (D835). We analysed 24 myeloid sarcoma specimens from 20 patients for FLT3 ITD and D835 mutations. FLT3 ITD mutations were identified in three of 20 cases (15%); no D835 mutations were identified. The ITD inserts ranged in size from 33 to 198 base pairs (bp) and represented approximately 20–40% of the FLT3 alleles. Two cases showed discordance in FLT3 ITD mutational status. In one case, the leukaemia specimen was positive for a FLT3 ITD mutation and the myeloid sarcoma specimen was negative. In the second case, the myeloid sarcoma was positive for a FLT3 ITD mutation at diagnosis, but negative in subsequent relapse samples. Our findings suggest that small molecule inhibitors of FLT3 may be useful therapeutic agents for treatment of myeloid sarcomas‐containing FLT3 mutations, however, the potential for discordance between the leukaemia and myeloid sarcoma, necessitates that the myeloid sarcoma tumour itself be analysed for FLT3 mutations.
Dual targeting of Bruton's tyrosine kinase and Janus kinase 3 with rationally designed inhibitors prevents graft‐versus‐host disease (GVHD) in a murine allogeneic bone marrow transplantation model
Authors: Cetkovic‐Cvrlje Marina , Uckun Fatih M.
Published: October 2004

The purpose of the present study was to evaluate the effectiveness of targeting Bruton's tyrosine kinase (BTK) with a specific BTK inhibitor, α‐cyano‐β‐hydroxy‐β‐methyl‐N‐(2,5‐dibromophenyl)‐propenamide (LFM‐A13), for prevention of acute fatal graft‐versus‐host disease (GVHD) in a murine model of allogeneic bone marrow transplantation (BMT). Vehicle‐treated control C57BL/6 mice receiving bone marrow/splenocyte grafts from allogeneic BALB/c donors developed severe multi‐organ acute GVHD and died after a median survival time (MST) of 40 d. LFM‐A13 treatment (25 mg/kg/d) significantly prolonged the MST of the BMT recipients to 47 d. The probability of survival at 2 months after BMT was 2 ± 2% for vehicle‐treated control mice and 22 ± 6% for mice treated with LFM‐A13 (P = 0·0008). Notably, the combination regimen of LFM‐A13 plus the standard anti‐GVHD drug methotrexate (MTX) (10 mg/m2/d) was more effective than LFM‐A13 alone, while the combination regimen of LFM‐A13 plus the novel anti‐GVHD drug JANEX‐1 (60 mg/kg/d), targeting Janus kinase 3, was more effective than LFM‐A13, JANEX‐1 or MTX alone. More than 70% of recipients receiving this most effective GVHD prophylaxis (LFM‐A13 + JANEX‐1) remained alive throughout the 80‐d observation period with an MST of >80 d. Taken together, these results indicate that targeting BTK with the chemical inhibitor LFM‐A13 may attenuate the severity of GVHD, especially when it is combined with other anti‐GVHD drugs, such as MTX and JANEX‐1.
Impaired generation of bone marrow CD34‐derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome
Authors: Micheva Ilina , Thanopoulou Eleni , Michalopoulou Sotiria , Kakagianni Theodora , Kouraklis‐Symeonidis Alexandra , Symeonidis Argiris , Zoumbos Nicholas
Published: October 2004

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34+progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine‐supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34+progenitors had low potential to generate DCsin vitro, as the number of DCs obtained from one CD34+cell was significantly lower compared with controls (median value 0·2 vs. 4,P = 0·003). In patients, the survival and proliferation of CD34+cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34+‐derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0·10 ± 0·10% vs. 0·35 ± 0·13%,P < 0·001; plasmacytoid DC: 0·11 ± 0·10% vs. 0·37 ± 0·14%,P < 0·001). In cases with the 5q deletion both CD34‐derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective ‘dendritopoiesis’ with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.
Circulating reticulated platelets in the early and late phases after ischaemic stroke and transient ischaemic attack
Authors: McCabe Dominick J. H. , Harrison Paul , Sidhu Paul S. , Brown Martin M. , Machin Samuel J.
Published: October 2004

The percentage of reticulated platelets (% RP) could be a useful marker of increased platelet production and/or turnover in patients with increased platelet activation, but few flow cytometric studies have measured the % RP in patients with ischaemic cerebrovascular disease (CVD). Whole blood flow cytometry using thiazole orange was performed to compare the % RP in patients in the early (1–27 d, n = 79) and late phases (79–725 d, n = 70) after ischaemic stroke or transient ischaemic attack (TIA) with controls without CVD (n = 27). The impact of aspirin dose escalation (75–300 mg/d) on the % RP was investigated in 10 patients in the late phase after stroke/TIA. The platelet count and mean platelet volume (MPV) were similar in CVD patients and controls. Compared with controls, the unadjusted % RP was not significantly higher in early or late phase CVD patients (P ≤ 0·3). However, having adjusted for age, the % RP was higher in early (P = 0·047) and late phase CVD patients (P = 0·01). There was a positive correlation between % RP and MPV in EDTA‐ and citrate‐anticoagulated blood in both early and late phase CVD patients (P≤ 0·01). The % RP was not significantly influenced by aspirin dose. These data do not convincingly support an excessive stimulus to platelet production in the early or late phases after ischaemic stroke/TIA, but are consistent with the hypothesis that reticulated platelets are larger than more mature ‘non‐reticulated’ platelets in ischaemic CVD.
Book review
Authors: Martin Gael M.
Published: October 2004

Bayesian Econometrics By Gary Koop.
Refractory status epilepticus due to acute hepatic porphyria in a pregnant woman: induced abortion as the sole therapeutic option?
Authors: Engelhardt K. , Trinka E. , Franz G. , Unterberger I. , Spiegel M. , Beer R. , Pfausler B. , Kampfl A. , Schmutzhard E.
Published: October 2004

A 22‐years old, 55 kg female patient in the twelfth week of pregnancy developed neuropsychiatric syndromes and in the following status epilepticus. Raised porphyrines and porphyrine precursors were found in the patient's urine. Despite intravenous glucose infusions and appropriate medication no reduction in seizure‐frequency and neuropsychiatric syndromes was observed. An abortion was induced. After the interruption and starting of haem arginate therapy, seizure activity stopped and porphyrine precursors returned to normal levels, and after 6 weeks the patient was discharged in excellent clinical condition. This report describes a status epilepticus caused by acute hepatic porphyria, triggered by pregnancy, in a 22‐years old woman. To our knowledge this is the first report of induced abortion as successful treatment in acute hepatic porphyria induced status epilepticus.
Progressive dementia caused by Hashimoto's encephalopathy – report of two cases
Authors: Spiegel J. , Hellwig D. , Becker G. , Müller M.
Published: October 2004

Dementia induced by Hashimoto's encephalopathy (HE) seems to be a rare condition. We report on two patients, who revealed a syndrome consisting of a rapid progressive dementia with myocloni. In both patients, the detection of antithyroid antibodies enabled the diagnosis of HE. The symptoms receded completely during a high‐dose glucocorticoid therapy. In patients with rapidly progressive dementia or with dementia of unknown origin, HE should be considered.
Two subduction events in a polycyclic basement: Alpine and pre‐Alpine high‐pressure metamorphism in the Suretta nappe, Swiss Eastern Alps
Authors: NUSSBAUM C. , MARQUER D. , BIINO G. G.
Published: October 2004

This study is essentially based on coupling macrostructures, microstructures and metamorphic petrology in polymetamorphic mafic rocks from the Swiss Eastern Alps (Suretta nappe, Penninic domain). Petrographic criteria are used in conjunction with structural analysis and microprobe work to define crystallization/deformation relationships and to establish a relative but precise sequence of tectono‐metamorphic events. A first eclogite facies overprint and related exhumation occurred before emplacement of late Palaeozoic intrusives. During the Alpine cycle, the Suretta nappe was part of the thinned European continental margin. The Tertiary burial due to subduction and collision is responsible for D1 ductile thrusting and blueschist facies metamorphism. Late deformation phases, related to exhumation, are responsible for the development of extensional structures under greenschist facies conditions. Quantitative metamorphic petrology based on Gibbs free energy minimization (DOMINO by de Capitani) gives a constraint on theP–T conditions during the polymetamorphic and polycyclic evolution. The first high‐Pmetamorphic event related to pre‐Alpine structures occurred atc. 700 °C and at least 2.0 GPa. These conditions are compatible with pre‐Alpine high‐Pre‐equilibration already described in several Alpine units. The Alpine high‐Pmetamorphism occurred under blueschist facies conditions atc. 400–450 °C and 1.0 GPa. Similar high‐P, low‐T conditions have already been described in the Mesozoic and Permian rock types. The two high‐Pevents are clearly related to two different geothermal regimes and geodynamic environments.
Diffusion‐controlled olivine corona textures in granitic rocks from Lofoten, Norway: calculation of Onsager diffusion coefficients, thermodynamic modelling and petrological implications
Authors: MARKL G. , FOSTER C. T. , BUCHER K.
Published: October 2004

Reactions occurring during cooling of charnockitic intrusives on the Lofoten Islands produce characteristic diffusion‐controlled textures around fayalite and Fe–Ti oxides. Thermobarometry indicates the corona textures formed at 780–840 °C and pressures of 4–10 kbar, whereas the magmatic assemblage of the charnockite (clinopyroxene–olivine–quartz) crystallized at about 850–870 °C and 4 kbar. The succession olivine|orthopyroxene+magnetite|orthopyroxene+garnet and olivine|orthopyroxene+magnetite|amphibole developed where olivine reacted with adjacent plagioclase or K‐feldspar, but the modes and the thicknesses of the corona textures vary according to the feldspar type, indicating that the primary magmatic ternary feldspar was already exsolved into albitic plagioclase and alkali feldspar when the corona formation began. Simultaneously, in other parts of the rock, primary magmatic clinopyroxene reacted to amphibole and Fe–Ti oxides reacted to orthopyroxene+garnet coronas or to amphibole. Textures demonstrate significant Al diffusion in the rocks under granulite facies conditions and they suggest that no pervasive fluid influx occurred and that amphibole formation was dependant on a local source of H2O probably related to water‐release during the last stages of magmatism. Calculation of the net reaction by accounting for all observed reactions at different sites in the rock indicates that the system can be regarded as balanced on a hand‐specimen scale with respect to all elements except for Na and H2O. The larger variety of textures developed in rocks of granitic bulk composition provide more constraints than textures from gabbroic compositions, and permitted calculation of a set of relative diffusion coefficients which also reproduce textures in the gabbroic and anorthositic rocks from the Lofoten Islands. The following set of relative diffusion coefficients (Li/LFe) reproduces the observed textures in the Lofoten rocks: Si=0.82, Mg=0.59, Mn=0.05, Na=0.38, K=0.39, Al=0.05 and Ca=0.07.
High‐pressure–low‐temperature metamorphism and deformation in the Bündnerschiefer of the Engadine window: implications for the regional evolution of the eastern Central Alps
Authors: BOUSQUET* R. , OBERHÄNSLI R. , GOFFE B. , JOLIVET L. , VIDAL O.
Published: October 2004

A study of the metamorphic and tectonic evolution of the Bündnerschiefer of the Engadine window shows that the individual nappes have been thinned by a large amount and that extension was active during and soon after nappe stacking.

Based on contrasting P–T  histories the Penninic Bündnerschiefer can be divided in two major units bounded by a horizontal contact. The lower (Mundin) unit shows typical high‐P/low‐T  parageneses in metapelites (Mg‐carpholite) and in metabasites (glaucophane); metamorphic conditions are estimated around 12 kbar, 375 °C. The upper (Arina) unit contains no specific high‐P minerals; metamorphic conditions are estimated around 7 kbar, 325 °C. A minimum pressure gap of 5 kbar is thus observed. The contact between the two units is marked by a mappable normal shear zone with top‐to‐the‐north‐west sense of shear. Near the shear zone, fresh carpholite fibres trend parallel to the regional stretching lineation, implying that the detachment is an early structure active from the depth of stability of the carpholite and persisting during subsequent exhumation. The good preservation of carpholite and the absence of retrograde chloritoid below the shear zone show that exhumation occurred along a cooling path, whereas the deeper units are exhumed along an isothermal path. Exhumation probably occurred during convergence and further nappe stacking during the earlier Eocene. These results suggest that pre‐collisional tectonic thinning of the Penninic oceanic units may be more widespread and significant than generally recognized.
Effects of cation substitutions in garnet and pyroxene on equilibrium oxygen isotope fractionations
Authors: KOHN M. J. , VALLEY J. W.
Published: October 2004

High‐grade metamorphic rocks were used to explore oxygen isotope fractionations between pyroxene and garnet, and to investigate the effects on fractionation factors of the cation substitutions Fe3+Al−1 and Ca(Fe,Mg)−1. Recrystallized, granulite facies (725 °C) wollastonite ores from the northern Adirondack highlands contain essentially only the minerals clinopyroxene (a Di–Hd solid solution)+garnet (a Grs–Adr solid solution)±wollastonite, and exhibit a systematic dependence of measured fractionations on the Fe3+ content of calcic garnet: Δ(Cpx–CaGrt)=(0.14±0.12)+(0.78±0.20)XAdr and Δ(Wo–CaGrt)=(0.15±0.22)+(0.57±0.33)XAdr. In eclogites formed at T ≤650 °C, measured compositions of Ca‐poor garnet and omphacite combined with experimental data indicate that Ca‐poor, Fe‐rich garnet is enriched in 18O compared to both diopside and grossular: extrapolating to 1000 K, Δ(Alm–Di)≈c. 0.2 and Δ(Alm–Grs)≈c. 0.5. Orthopyroxene and clinopyroxene from Gore Mountain, New York, show a constant fractionation that is independent of rock type, as expected if they have the same closure temperature. These data imply Δ(Opx‐Cpx)≈c. 0.7 at 1000 K. Measured fractionations among Ca‐poor garnet, orthopyroxene, clinopyroxene and hornblende in the Gore Mountain rocks further indicate an 18O enrichment in Ca‐poor garnet over Grs (≈c. 0.5 at 1000 K). The new measurements are indistinguishable from expected equilibrium values based on experiments for the minerals enstatite, diopside, grossular, wollastonite and feldspar, but consistently indicate a significant isotope effect for the simple octahedral cation substitutions Fe3+Al−1 (Grs vs. Adr) and Ca(Fe,Mg)−1 (Ca‐poor garnet vs. Grs; Opx vs. Cpx). Neither cation substitution has been directly investigated for its effect on 18O/16O fractionation with experiments in silicates. Chemical characterization of minerals is required prior to petrological interpretation of oxygen isotope trends.
Episodic porphyroblast growth in the Fleur de Lys Supergroup, Newfoundland: timing relative to the sequential development of multiple crenulation cleavages
Authors: STALLARD A.
Published: October 2004

Inclusion trails in garnet and albite porphyroblasts in the Fleur de Lys Supergroup preserve successive generations of microstructures, some of which correlate with equivalent microstructures in the matrix. Microstructure–porphyroblast relationships provide timing constraints on a succession of seven crenulation cleavages (S1–S7) and five stages of porphyroblast growth. Significant destruction and alteration of early fabrics has occurred during the microstructural development of the rock mass. Garnet porphyroblasts grew episodically through four growth stages (G1–G4) and preserve a succession of five fabrics (S1–S5) as inclusion trails. Garnet growth during each of the four growth phases did not occur on all pre‐existing porphyroblasts, resulting in contrasting growth histories between individual garnet porphyroblasts from the same outcrop. Albite porphyroblasts grew during a single stage of growth and have overgrown microstructures continuous with the matrix. The garnet and albite porphyroblast inclusion trails record a succession of crenulation cleavages without any rotation of the porphyroblasts relative to other porphyroblasts in the population.

Complex microstructural histories are best resolved by preparing multiple oriented thin sections from a large number of samples of different rock types within the area of study. The succession of matrix foliations must be understood, as it provides the most useful time‐frame against which to measure the relative timing of phases of porphyroblast growth. Comparable microstructures must be identified in different porphyroblasts and in the rock matrix.
Crustal thickening of the lower crust of the Kohistan arc (N. Pakistan) deduced from Al zoning in clinopyroxene and plagioclase
Authors: YOSHINO* T. , YAMAMOTO H. , OKUDAIRA T. , TORIUMI M.
Published: October 2004

The lower‐crustal rocks of the Kohistan complex (northern Pakistan) are mostly composed of metabasic rocks such as pyroxene granulites, garnet granulites and amphibolites. We have investigated P–T  trajectories of the relic two‐pyroxene granulites, which are the protolith of the amphibolites within the Kamila amphibolite belt. Aluminous pyroxene retains igneous textures such as exsolution lamellae developed in the core. The significant amount of Al in clinopyroxene is buffered by breakdown reactions of plagioclase accompanied by film‐like quartz as a product at grain boundaries between plagioclase and clinopyroxene. Distinct Al zoning profiles are preserved in pyroxene with exsolution lamellae in the core and in plagioclase adjacent to clinopyroxene in pyroxene granulites. In the northern part of the Kamila amphibolite belt, Al in clinopyroxene increases towards the rim and abruptly decreases at the outer rim, and anorthite in plagioclase decreases towards the rim and abruptly increases near the grain boundary between plagioclase and clinopyroxene. In the southern part of the Kamila amphibolite belt, Al in clinopyroxene and anorthite in plagioclase simply increase towards the margins of the grains. The anorthite zoning in plagioclase is in agreement with the zoning profiles of Ca‐Tschermaks and jadeite components inferred from variations of Al, Na, Ti and Fe3+ in clinopyroxene. Assuming that the growth surface between them was in equilibrium, geothermobarometry based on Al zoning in clinopyroxene coexisting with plagioclase indicates that metamorphic pressures significantly increased with increasing temperature under granulite facies metamorphism. The peak of granulite facies metamorphism occurred at conditions of about 800 °C and 800–1100 MPa. These prograde P–T  paths represent a crustal thickening process of the Kohistan arc during the Early to Middle Cretaceous. The crustal thickening of the Kohistan arc was caused by accretion of basaltic magma at mid‐crustal depths.
Stable isotopic evidence for fluid infiltration during contact metamorphism in a multiply‐metamorphosed terrane: the Reynolds Range, Arunta Block, central Australia
Authors: VRY J. K. , CARTWRIGHT I.
Published: October 2004

The Lander Rock Beds form the local basement of the Reynolds Range in the Arunta Inlier of central Australia. These dominantly quartzose and pelitic lithologies underwent low‐grade (c. 400 °C) regional metamorphism prior to contact metamorphism (c. 2.5 kbar) around S‐type megacrystic granitoids at 1820–1800 Ma. The Lander Rock Beds are overlain by metasediments of the Reynolds Range Group, which were subsequently intruded by granitoids at c. 1780 Ma. Regional metamorphism at 1590–1580 Ma produced grades varying from greenschist (400 °C at 4–5 kbar) to granulite (750–800 °C at 4–5 kbar) from north‐west to south‐east along the length of the Reynolds Range. Oxygen isotope ratios of the Lander Rock Beds were reset from 13.4±0.8 to as low as 6.7 adjacent to the contacts of the larger plutons, and to 10.3±1.1 around the smaller plutons. Biotite in all the major rock types found in the aureoles has δD values between −52 and −69, probably reflecting resetting by a cooling igneous+metamorphic fluid near the plutons. Sapphirine‐bearing and other Mg‐ and Al‐rich rock types have low δ18O values (4.0±0.7). The precursors to these rocks were probably low‐temperature (c. 200 °C) diagenetic–hydrothermal deposits of Mg‐rich chlorite, analogous to those in Proterozoic stratiform precious metal and uranium deposits that form by the infiltration of basin brines or seawater. As in the overlying Reynolds Range Group, regional metamorphism involved little fluid–rock interaction and isotopic resetting.
Do Phonological and Executive Processes in English Learners at Risk for Reading Disabilities in Grade 1 Predict Performance in Grade 2?
Authors: Lee Swanson H. , Sáez Leilani , Gerber Michael
Published: October 2004

This study determined the degree to which the phonological and executive components of memory reflect language‐specific capacities in reading achievement. We tested whether the memory processes in a sample of English‐language learners that played a major role in predicting second‐language acquisition and risk for reading disability (RD) in Grade 1 (Swanson, Sáez, Gerber, & Leafstedt, 2004) also predicted reading performance in Grade 2. The present results showed that Spanish short‐term memory (STM) performance in Grade 1 predicted basic Spanish‐reading skills and Spanish comprehension in Grade 2, whereas Grade 1 English STM performance predicted English vocabulary and English comprehension in Grade 2. More importantly, children at risk for RD in Grade 1 differed from the counterparts in Grade 2 on both English and Spanish measures of reading, whereas their memory deficits were isolated to Spanish STM and working memory (WM). The relationship between language‐specific processes in memory and reading are discussed.
English Reading Effects of Small‐Group Intensive Intervention in Spanish for K–1 English Learners
Authors: Gerber Michael , Jimenez Terese , Leafstedt Jill , Villaruz Jessica , Richards Catherine , English Judy
Published: October 2004

In this article we report small, but statistically significant, effects of brief supplemental instruction on English reading by Spanish‐speaking kindergartners (N = 37) who performed poorly on a bilingual battery of phonological‐processing tasks. Intervention design was compatible with the Reading First initiative and with research on use of multitiered intervention strategies for preventing reading failure among young monolingual students (e.g., L. S. Fuchs & Vaughn, 2003). We describe a Core Intervention Model (CIM) comprised of specific instructional behaviors that teachers might easily learn and employ regardless of curriculum, and discuss implications of our findings for building multitiered preventative instruction for young English learners.
Effectiveness of Explicit Phonological‐Awareness Instruction for At‐Risk English Learners
Authors: Leafstedt Jill M. , Richards Catherine R. , Gerber Michael M.
Published: October 2004

This article examines the effects of intensive phonological‐awareness (PA) instruction for kindergarten English learners. One intact kindergarten class was provided 300 minutes of intensive instruction in PA. Results indicate that students who received intervention made significant growth in word reading when compared to a cohort of kindergarten students who received general kindergarten instruction. The article also discusses ability‐group differences in performance among high‐, middle‐, and low‐performing students and the implications of these findings for instructional practice.
Use of Semiconductor Quantum Dots for Photostable Immunofluorescence Labeling of
Cryptosporidium parvum
Authors: Lee Lai Yoke , Ong Say Leong , Hu Jiang Yong , Ng Wun Jern , Feng Yaoyu , Tan Xiaolan , Wong Shih Wei
Published: October 2004

Cryptosporidium parvum
is a waterborne pathogen that poses potential risk to drinking water consumers. The detection of
Cryptosporidium
oocysts, its transmissive stage, is used in the latest U.S. Environmental Protection Agency method 1622, which utilizes organic fluorophores such as fluorescein isothiocyanate (FITC) to label the oocysts by conjugation with anti-
Cryptosporidium
sp. monoclonal antibody (MAb). However, FITC exhibits low resistance to photodegradation. This property will inevitably limit the detection accuracy after a short period of continuous illumination. In view of this, the use of inorganic fluorophores, such as quantum dot (QD), which has a high photobleaching threshold, in place of the organic fluorophores could potentially enhance oocyst detection. In this study, QD605-streptavidin together with biotinylated MAb was used for
C. parvum
oocyst detection. The
C. parvum
oocyst detection sensitivity increased when the QD605-streptavidin concentration was increased from 5 to 15 nM and eventually leveled off at a saturation concentration of 20 nM and above. The minimum QD605-streptavidin saturation concentration for detecting up to 4,495 ± 501 oocysts (mean ± standard deviation) was determined to be 20 nM. The difference in the enumeration between 20 nM QD605-streptavidin with biotinylated MAb and FITC-MAb was insignificant (
P
> 0.126) when various
C. parvum
oocyst concentrations were used. The QD605 was highly photostable while the FITC intensity decreased to 19.5% ± 5.6% of its initial intensity after 5 min of continuous illumination. The QD605-based technique was also shown to be sensitive for oocyst detection in reservoir water. This observation showed that the QD method developed in this study was able to provide a sensitive technique for detecting
C. parvum
oocysts with the advantage of having a high photobleaching threshold.
Distribution of
Escherichia coli
O157 in Bovine Fecal Pats and Its Impact on Estimates of the Prevalence of Fecal Shedding
Authors: Pearce M. C. , Fenlon D. , Low J. C. , Smith A. W. , Knight H. I. , Evans J. , Foster G. , Synge B. A. , Gunn G. J.
Published: October 2004

The distribution of
Escherichia coli
O157 in bovine feces was examined by testing multiple samples from fecal pats and determining the density of
E. coli
O157 in immunomagnetic separation (IMS)-positive fecal samples. The density of
E. coli
O157 in bovine feces was highly variable, differing by as much as 76,800 CFU g
−1
between samples from the same fecal pat. The density in most positive samples was <100 CFU g
−1
, the limit of reliable detection by IMS. Testing only one 1-g sample of feces per pat with IMS may result in a sensitivity of detection as low as 20 to 50%. It is therefore probable that most surveys have greatly underestimated the prevalence of
E. coli
O157 shedding in cattle and the proportion of farms with shedding cattle. The sensitivity of the detection of
E. coli
O157 in bovine feces can be as much as doubled by testing two 1-g samples per pat rather than one 1-g sample.
Mechanisms of Bactericidal Action of Cinnamaldehyde against
Listeria monocytogenes
and of Eugenol against
L. monocytogenes
and
Lactobacillus sakei
Authors: Gill Alexander O. , Holley Richard A.
Published: October 2004

The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure. In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to
Listeria monocytogenes
. At a concentration of 6 mM eugenol was bactericidal to
Lactobacillus sakei
, but treatment with 0.5 M cinnamaldehyde had no significant effect. To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured. Treatment of nonenergized
L. monocytogenes
with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 μM carbonyl cyanide
m
-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose. Treatment of energized
L. monocytogenes
with 40 mM cinnamaldehyde or 10 μM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP. Treatment of
L. sakei
with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells. CCCP at a concentration of 100 μM had no significant effect on the cellular ATP of
L. sakei
. No significant changes in extracellular ATP were observed. Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations. The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability.
Stable-Isotope Probing of Microorganisms Thriving at Thermodynamic Limits: Syntrophic Propionate Oxidation in Flooded Soil
Authors: Lueders Tillmann , Pommerenke Bianca , Friedrich Michael W.
Published: October 2004

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H
2
-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [
13
C]propionate (<10 mM) and the oxidation of ∼30 μmol of
13
C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were
13
C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed
Syntrophobacter
spp.,
Smithella
spp., and the novel
Pelotomaculum
spp. to predominate in “heavy”
13
C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the
Archaea
, mostly
Methanobacterium
and
Methanosarcina
spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of
13
C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [
13
C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.
Lactate-Utilizing Bacteria, Isolated from Human Feces, That Produce Butyrate as a Major Fermentation Product
Authors: Duncan Sylvia H. , Louis Petra , Flint Harry J.
Published: October 2004

The microbial community of the human colon contains many bacteria that produce lactic acid, but lactate is normally detected only at low concentrations (<5 mM) in feces from healthy individuals. It is not clear, however, which bacteria are mainly responsible for lactate utilization in the human colon. Here, bacteria able to utilize lactate and produce butyrate were identified among isolates obtained from 10
−8
dilutions of fecal samples from five different subjects. Out of nine such strains identified, four were found to be related to
Eubacterium hallii
and two to
Anaerostipes caccae
, while the remaining three represent a new species within clostridial cluster XIVa based on their 16S rRNA sequences. Significant ability to utilize lactate was not detected in the butyrate-producing species
Roseburia intestinalis
,
Eubacterium rectale
, or
Faecalibacterium prausnitzii
. Whereas
E. hallii
and
A. caccae
strains used both
d
- and
l
-lactate, the remaining strains used only the
d
form. Addition of glucose to batch cultures prevented lactate utilization until the glucose became exhausted. However, when two
E. hallii
strains and one
A. caccae
strain were grown in separate cocultures with a starch-utilizing
Bifidobacterium adolescentis
isolate, with starch as the carbohydrate energy source, the
l
-lactate produced by
B. adolescentis
became undetectable and butyrate was formed. Such cross-feeding may help to explain the reported butyrogenic effect of certain dietary substrates, including resistant starch. The abundance of
E. hallii
in particular in the colonic ecosystem suggests that these bacteria play important roles in preventing lactate accumulation.
Identification of the Receptor-Binding Protein in 936-Species Lactococcal Bacteriophages
Authors: Dupont Kitt , Vogensen Finn Kvist , Neve Horst , Bresciani José , Josephsen Jytte
Published: October 2004

The aim of this work was to identify genes responsible for host recognition in the lactococcal phages sk1 and bIL170 belonging to species 936. These phages have a high level of DNA identity but different host ranges. Bioinformatic analysis indicated that homologous genes,
orf18
in sk1 and
orf20
in bIL170, could be the receptor-binding protein (RBP) genes, since the resulting proteins were unrelated in the C-terminal part and showed homology to different groups of proteins hypothetically involved in host recognition. Consequently, chimeric bIL170 phages carrying
orf18
from sk1 were generated. The recombinant phages were able to form plaques on the sk1 host
Lactococcus lactis
MG1614, and recombination was verified by PCR analysis directly with the plaques. A polyclonal antiserum raised against the C-terminal part of phage sk1 ORF18 was used in immunogold electron microscopy to demonstrate that ORF18 is located at the tip of the tail. Sequence analysis of corresponding proteins from other lactococcal phages belonging to species 936 showed that the N-terminal parts of the RBPs were very similar, while the C-terminal parts varied, suggesting that the C-terminal part plays a role in receptor binding. The phages investigated could be grouped into sk1-like phages (p2, fd13, jj50, and φ7) and bIL170-like phages (P008, P113G, P272, and bIL66) on the basis of the homology of their RBPs to the C-terminal part of ORF18 in sk1 and ORF20 in bIL170, respectively. Interestingly, sk1-like phages bind to and infect a defined group of
L. lactis
subsp.
cremoris
strains, while bIL170-like phages bind to and infect a defined group of
L. lactis
subsp.
lactis
strains.
Identification of
Lactococcus lactis
Genes Required for Bacteriophage Adsorption
Authors: Dupont Kitt , Janzen Thomas , Vogensen Finn Kvist , Josephsen Jytte , Stuer-Lauridsen Birgitte
Published: October 2004

The aim of this work was to identify genes in
Lactococcus lactis
subsp.
lactis
IL1403 and
Lactococcus lactis
subsp.
cremoris
Wg2 important for adsorption of the 936-species phages bIL170 and φ645, respectively. Random insertional mutagenesis of the two
L. lactis
strains was carried out with the vector pGh9:IS
S1
, and integrants that were resistant to phage infection and showed reduced phage adsorption were selected. In
L. lactis
IL1403 integration was obtained in the
ycaG
and
rgpE
genes, whereas in
L. lactis
Wg2 integration was obtained in two genes homologous to
ycbC
and
ycbB
of
L. lactis
IL1403.
rgpE
and
ycbB
encode putative glycosyltransferases, whereas
ycaG
and
ycbC
encode putative membrane-spanning proteins with unknown functions. Interestingly,
ycaG
,
rgpE
,
ycbC
, and
ycbB
are all part of the same operon in
L. lactis
IL1403. This operon is probably involved in biosynthesis and transport of cell wall polysaccharides (WPS). Binding and infection studies showed that φ645 binds to and infects
L. lactis
Wg2,
L. lactis
IL1403, and
L. lactis
IL1403 strains with pGh9:IS
S1
integration in
ycaG
and
rgpE
, whereas bIL170 binds to and infects only
L. lactis
IL1403 and cannot infect Wg2. These results indicate that φ645 binds to a WPS structure present in both
L. lactis
IL1403 and
L. lactis
Wg2, whereas bIL170 binds to another WPS structure not present in
L. lactis
Wg2. Binding of bIL170 and φ645 to different WPS structures was supported by alignment of the receptor-binding proteins of bIL170 and φ645 that showed no homology in the C-terminal part.
Functional Analysis of Fructosyl-Amino Acid Oxidases of
Aspergillus oryzae
Authors: Akazawa Shin-ichi , Karino Tetsuya , Yoshida Nobuyuki , Katsuragi Tohoru , Tani Yoshiki
Published: October 2004

Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from
Aspergillus oryzae
strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward
N
ε
-fructosyl
N
α
-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e.,
FAOAo1
and
FAOAo2
) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in
Escherichia coli
and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active.
FAOAo2
disruptant was obtained by using
ptrA
as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain
ΔfaoAo2
did not grow. Addition of glucose or (NH
4
)
2
SO
4
to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the
FAOAo2
gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO
2
as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in
A. oryzae
.
Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism inPichia anomala
Authors: Fredlund Elisabeth , Blank Lars M. , Schnürer Johan , Sauer Uwe , Passoth Volkmar
Published: October 2004

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyceswine yeastPichia anomala. In aerobic batch culture,P. anomalagrows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose−1) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate thatP. anomalaexhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.
Evaluation of Methods for Storage of Marine Macroorganisms with Optimal Recovery of Bacteria
Authors: Siebert Kathrin , Busl Martina , Asmus Irina , Freund Josef , Muscholl-Silberhorn Albrecht , Wirth Reinhard
Published: October 2004

Marine macroorganisms are a potential source for new bioactive substances. In many cases marine microorganisms—especially bacteria—associated with these macroorganisms are actually producing the bioactive substances. One often is not able to immediately isolate microorganisms from collected macroorganismic materials; we therefore evaluated different methods for storage of such material, e.g., on board research vessels. These methods were the following: storage of macerates in sintered glass beads and 5% trehalose at −20°C (SGT method); storage of sections in 5% dimethyl sulfoxide at −70°C (SD method); storage of macerates at −20°C using the commercial ROTI-STORE system (RS method); storage of macerates at −20°C in 50% glycerol (GC method); and storage of macerates covered by mineral oil at 4°C (MO method). The SGT and SD methods resulted in numbers of and especially diversity of recoverable bacteria that were higher than for the other methods. Data for the RS method indicated its potential usefulness, too. The MO method resulted in growth during storage, thereby enriching a few selected microorganisms; the GC method resulted in a survival and diversity of recovered bacteria that was too low.
Functional Expression of
Sinorhizobium meliloti
BetS, a High-Affinity Betaine Transporter, in
Bradyrhizobium japonicum
USDA110
Authors: Boscari Alexandre , Mandon Karine , Poggi Marie-Christine , Le Rudulier Daniel
Published: October 2004

Among the
Rhizobiaceae
,
Bradyrhizobium japonicum
strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of
B. japonicum
, cells were transformed with the
betS
gene of
Sinorhizobium meliloti
. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na
+
/H
+
antiporters in
B. japonicum
could explain its very high Na
+
sensitivity.
Vulnerability of Drinking-Water Wells in La Crosse, Wisconsin, to Enteric-Virus Contamination from Surface Water Contributions
Authors: Borchardt Mark A. , Haas Nathaniel L. , Hunt Randall J.
Published: October 2004

Human enteric viruses can contaminate municipal drinking-water wells, but few studies have examined the routes by which viruses enter these wells. In the present study, the objective was to monitor the municipal wells of La Crosse, Wisconsin, for enteric viruses and determine whether the amount of Mississippi River water infiltrating the wells was related to the frequency of virus detection. From March 2001 to February 2002, one river water site and four wells predicted by hydrogeological modeling to have variable degrees of surface water contributions were sampled monthly for enteric viruses, microbial indicators of sanitary quality, and oxygen and hydrogen isotopes.
18
O/
16
O and
2
H/
1
H ratios were used to determine the level of surface water contributions. All samples were collected prior to chlorination at the wellhead. By reverse transcription-PCR (RT-PCR), 24 of 48 municipal well water samples (50%) were positive for enteric viruses, including enteroviruses, rotavirus, hepatitis A virus (HAV), and noroviruses. Of 12 river water samples, 10 (83%) were virus positive by RT-PCR. Viable enteroviruses were not detected by cell culture in the well samples, although three well samples were positive for culturable HAV. Enteroviruses detected in the wells by RT-PCR were identified as several serotypes of echoviruses and group A and group B coxsackieviruses. None of the well water samples was positive for indicators of sanitary quality, namely male-specific and somatic coliphages, total coliform bacteria,
Escherichia coli
, and fecal enterococci. Contrary to expectations, viruses were found in all wells regardless of the level of surface water contributions. This result suggests that there were other unidentified sources, in addition to surface water, responsible for the contamination.