bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection of hematopoietic stem cells with a combination of galactose‐bound vinyl polymer and soybean agglutinin, a galactose‐specific lectin
Authors: Yura Hirofumi , Kanatani Yasuhiro , Ishihara Masayuki , Takase Bonpei , Nambu Masaki , Kishimoto Satoko , Kitagawa Michihiro , Tatsuzawa Osamu , Hoshi Yasutaka , Suzuki Shinya , Kawakami Mitsuyuki , Matsui Takemi
Published: February 2008

BACKGROUND: Selection of hematopoietic stem cells can be used to prevent graft‐versus‐host disease (GVHD) after allograft transplantation. The purpose of the study was to examine a novel cell separation system comprising a galactose‐bound vinyl polymer (Gal‐VP) and soybean agglutinin (SBA), a galactose‐specific lectin.

STUDY DESIGN AND METHODS: A vinyl polymer (VP) containing α‐1,6‐ and β‐1,4‐linked galactose terminals was used to facilitate cell separation. A VP containing an α‐1,4‐linked glucose terminal (α‐1,4‐Glu‐VP) was also synthesized as a control for α‐1,6‐ and β‐1,4‐Gal‐VP. Peripheral blood samples were collected from healthy volunteers and umbilical cord blood cells were collected after normal labor.

RESULTS: The sugar‐VP was adsorbed on the surface of various materials. In the presence of SBA, T lymphocytes bound to β‐1,4‐Gal‐VP–coated microbeads, but not to α‐1,4‐Glu‐VP–coated microbeads. When peripheral or cord blood cells were cultured on α‐1,6‐Gal‐VP–coated plates, most red blood cells, lymphocytes, granulocytes, and monocytes adhered to the plate in the presence of 300 mg per mL SBA, whereas few CD34+ cells attached, even with 800 mg per mL SBA.

CONCLUSION: SBA binds selectively to blood cells by recognizing cell‐surface sugars, which are dependent on the extent of cellular differentiation. Therefore, the combination of α‐1,6‐Gal‐VP and SBA might be useful for separation of blood cells according to their stage of differentiation and lineage.
Evaluation of proposed FDA criteria for the evaluation of radiolabeled red cell recovery trials
Authors: Dumont Larry J. , AuBuchon James P. ,   Biomedical Excellence for Safer Transfusion (BEST) Collaborative
Published: February 2008

BACKGROUND: FDA requirements for recovery of radiolabeled red blood cells (RBCs) 24 hours after autologous reinfusion in normal subjects have evolved over time. This study defined the ability of currently available RBC systems to satisfy the most recently proposed criteria.

STUDY DESIGN METHODS: RBC recoveries were collected from US laboratories participating in clinical trials for RBC systems that have received FDA approval. Data were stratified for analysis into liquid‐stored, gamma‐irradiated, and frozen components. With statistical software, 24 individual samples were randomly selected with replacement from each stratum, repeating this for 5000 sample groups per stratum, to simulate experimental outcomes for each population. The percentage of sample groups that passed each and all of the proposed FDA criteria was determined. This procedure was repeated for recovery success thresholds of 75, 70, and 67 percent.

RESULTS: A total of 941 RBC recoveries were obtained from 11 laboratories and 34 studies performed between 1990 and 2006 for 12 sponsors. While the criterion for the mean was almost always satisfied, the standard deviation (SD) criterion was more problematic. Causing most failures was the success threshold definition. Changing the success threshold from 75 to 70 percent or 67 percent increased the likelihood of meeting the requirement for all RBC types.

CONCLUSION: The probability of passing the FDA‐proposed criteria for currently FDA‐approved products was poor. Changing the success threshold for an individual RBC recovery from 75 to 67 percent resulted in improved ability to meet this criterion for all three RBC types. This change had no affect on the pass rates based on the mean and SD criteria.
pH value promotes growth of Staphylococcus epidermidis in platelet concentrates
Authors: Störmer Melanie , Kleesiek Knut , Dreier Jens
Published: February 2008

BACKGROUND: The platelet (PLT) storage lesion is characterized metabolically by a pH value associated with lactic acid generation. PLT storage conditions support the growth of Staphylococcus epidermidis, the most common organism implicated in bacterial contamination of PLT concentrates (PCs). Here, different factors that influence bacterial growth in PCs are discussed and the relation between pH values of PCs and citrate plasma (CP) is studied, with emphasis on bacterial proliferation.

STUDY DESIGN AND METHODS: The PLT lesion with regard to pH decrease and lactic acid production was monitored during storage and correlated to bacterial proliferation properties. A total of 115 coagulase‐negative staphylococci, especially S. epidermidis isolates, were characterized for their proliferation in different blood components (CP, buffy coat–derived, and apheresis PCs). Furthermore, the influence of donor‐specific, product‐specific, species‐specific, and strain‐specific factors on bacterial proliferation was investigated.

RESULTS: PCs showed a lower pH value in comparison to plasma during storage. Bacterial proliferation in PCs and the failure to grow in CP were determined with all organisms tested. No correlation to donor‐specific, species‐specific, or strain‐specific factors was observed. Lowering the pH of CP resulted in bacterial proliferation, whereas a pH increase in the PC unit inhibited the proliferation of S. epidermidis.

CONCLUSION: With emphasis on bacterial proliferation, the significant difference between PC and CP is the presence of metabolizing PLTs. The pH values of stored PLTs, but not those of stored plasma, support the growth of S. epidermidis.
Proficiency testing experience for viable CD34+ stem cell analysis
Authors: López María C. , Lawrence David A.
Published: February 2008

BACKGROUND: Successful hematopoietic engraftment depends on the number of viable CD34+ stem cells. Therefore, accurate quantification of viable CD34+ stem cells is required.

STUDY DESIGN AND METHODS: To evaluate clinical laboratory performance, the New York State Department of Health initiated proficiency testing (PT) for viable CD34+ stem cells. Preserved adult peripheral blood was spiked with preserved cord blood CD34+ stem cells and was shipped to the participating laboratories. Three educational and two graded PTs were performed by participating laboratories, and their results were analyzed for consistency. Comparative analysis of viability with 7‐aminoactinomycin D (7‐AAD) and ToPro‐3 dyes also was performed.

RESULTS: Laboratories had to adapt their standard operating procedures to include a viability dye to quantify the number of viable CD34+ stem cells. The majority of laboratories chose 7‐AAD as their preferred viability dye, but propidium iodide (PI) and ToPro‐3 were used by two laboratories. Once all laboratories started to simultaneously analyze viability and staining for CD34, graded PTs started. Lower numbers of viable CD34+ stem cells were obtained for ToPro‐3 when the dye was compared with 7‐AAD.

CONCLUSION: It is concluded that ToPro‐3 stains more cells than 7‐AAD and likely includes compromised cells. The use of new vital dyes, like ToPro‐3, that may stain preapoptotic cells could represent an important advance to improve the quantification of viable CD34+ stem cells, for engraftment purposes. Further studies are needed to document the benefits of switching to a method that excludes not only dead cells, but apoptotic cells as well.
N‐terminal pro‐brain natriuretic peptide is a useful diagnostic marker for transfusion‐associated circulatory overload
Authors: Tobian Aaron A.R. , Sokoll Lori J. , Tisch Daniel J. , Ness Paul M. , Shan Hua
Published: February 2008

BACKGROUND: Transfusion‐associated circulatory overload (TACO) can be difficult to diagnose and distinguish from transfusion‐related acute lung injury (TRALI), but is a relatively common complication that occurs when increases in blood volume overwhelm the cardiovascular system. Brain natriuretic peptide (BNP) has been shown to be a functional marker for TACO. N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) is another marker that could be more helpful than BNP since it has a longer half‐life in circulation and is also much more stable in laboratory samples. In this study, whether NT‐proBNP is a useful diagnostic marker for TACO was evaluated.

STUDY DESIGN AND METHODS: Forty patients were enrolled into a case‐control study (16 patients with TACO and 24 control patients) and had pre‐ and posttransfusion NT‐proBNP concentrations evaluated from submitted type‐and‐screen blood samples.

RESULTS: The sensitivity of elevated posttransfusion NT‐proBNP to diagnose TACO was 93.8 percent, the specificity was 83.8 percent, and the accuracy was 87.5 percent. Elevated posttransfusion NT‐proBNP is the only independent variable for the diagnosis of TACO based on multivariate logistic regression.

CONCLUSION: NT‐proBNP is both a sensitive and a specific marker for TACO and can be helpful in confirming transfusion associated fluid overload. This study also demonstrates that many patients who experience TACO may already be in a state of excess volume. Clinicians should be aware that many asymptomatic patients have excess fluid and transfusion may cause these patients to become symptomatic.
Latitudinal Variation in Leaf and Tree Traits of the Mangrove Avicennia germinans (Avicenniaceae) in the Central Region of the Gulf of Mexico
Authors: Méndez‐Alonzo Rodrigo , López‐Portillo Jorge , Rivera‐Monroy Víctor H.
Published: February 2008

The variation in leaf mass per area, leaf nutrients (% carbon, nitrogen and phosphorus), and the allometric relation between tree height and diameter of the black mangrove, Avicennia germinans, were explored in nine mangrove forests in similar environments along a 5° latitudinal gradient in the central region of the Gulf of Mexico, as indicated by a southward increase in temperature and precipitation. There was no correlation between leaf nitrogen or phosphorus content and latitude. Leaf mass per area and leaf carbon content were positively correlated with latitude and negatively correlated with temperature and annual rainfall, whereas asymptotic tree height and maximum diameter showed the opposite trend. Such patterns suggest a trade‐off between leaf traits and tree size which may be constrained by the same environmental factors along a dry‐cold to humid‐warm latitudinal gradient.
Decline of Mammal Species Diversity Along the Yungas Forest of Argentina
Authors: Ojeda Ricardo A. , Barquez Rubén M. , Stadler Jutta , Brandl Roland
Published: February 2008

The southernmost extension of tropical Andean–Amazonian forests protrudes into the arid to semiarid habitats in northwestern Argentina. We analyzed the decline of species richness of forest mammals along these Yungas forests. In particular, we tested whether species decline is due to a general latitudinal effect and whether the drop of species from the assemblages was independent from species' traits and environmental variables. For these tests, we estimated the geographic range edges of 39 species of forest mammal and estimated the species richness with bands of 30′. First, we compared the slope of the decline of species richness from north to south (5.8 species/degree) with the decline expected from the latitudinal species gradient (1–3 species/degree). The decline in species richness of forest mammals along the Yungas was significantly steeper than expected. Second, with a null model assuming a random drop of mammal species we derived confidence limits for the expected species richness and number of range edges within the bands. None of the forest mammals reached the tip of the forest, in marked contrast to nonforest mammals. More range edges than expected from the null model fell within the bands 23°30′–24°00′ S and a band near the tip of the Yungas indicating a nonrandom drop of species. The correlation between vulnerability traits and range edges suggests that processes associated with the availability of resources influence the drop of species. We propose that a suite of macroecological attributes interacting with a decrease in habitat quality determines the pattern of species richness in the Yungas forest.
Long‐pollen Movement and Deviation of Random Mating in a Low‐density Continuous Population of a Tropical Tree Hymenaea courbaril in the Brazilian Amazon
Authors: Eduardo André , De Lacerda Biscaia , Kanashiro Milton , Sebbenn Alexandre Magno
Published: February 2008

Mating system and pollen flow are two key elements to understand the genetic structure of tree species. Mating and pollen‐dispersal patterns of a low‐density population of bat pollinated Hymanea courbaril were examined before logging in a 546‐ha plot in the Brazilian Amazon. The multilocus genotypes of nine microsatellite loci were determined for 130 adult‐trees and 367 seeds collected from 20 seed‐trees. Mating system analysis, using mixed‐mating model and paternity analysis showed that the studied population is perfectly outcrossed (tm= 1.002), and probably self‐incompatible. However, significant deviations from random mating were detected for mating among relatives (tm−ts= 0.096, P < 0.05) and correlated matings (rp= 0.289, P < 0.05), indicating inbreeding in the population and that part of offspring are full‐sibs (28.9%). Inbreeding was reflected in the positive and significant fixation index observed in adult trees (F= 0.137, P < 0.05), although no significant inbreeding was detected in offspring (F= 0.074, P > 0.05). The effective number of pollen donors mating with each seed‐tree was determined to be low (Nep≈ 4). The average of pollen flow distance was measured inside of the plot by both paternity (827 ± 429 m) and TwoGener analysis (115–363 m). However, this underestimated pollen dispersal distance, since the detected rate of pollen immigration inside of the plot was high (55%). The observed long‐pollen dispersal distance is probably related to pollination by bats and the low density of reproductive trees in the site.
Establishment of Epiphytic Bromeliads in Successional Tropical Premontane Forests in Costa Rica
Authors: Marín Alfredo Cascante , Wolf Jan H. D. , Oostermeijer J. Gerard B. , Den Nijs Joannes C. M.
Published: February 2008

Plant community composition is the combined result of species‐specific competitive abilities and the availability of propagules. For epiphytic plants, current hypotheses consider that dispersal‐related factors are most important. By controlling seed dispersal constraints, we experimentally examined whether the community composition of epiphytic bromeliads in a tropical premontane area is determined during early phases of seedling recruitment. Also, we tested whether establishment success was related to eco‐physiological traits of the species. A total of 7200 seeds were artificially affixed on several host trees in two secondary forest patches and in a mature forest stand. Four bromeliad species with differing physiological characteristics (CAM, C3‐CAM, and C3) and habitat preference (secondary vs. primary forest) were selected. We found that differences in seed germination probability among habitats and species were not likely to influence community assembly. After 2 yr, seedling survival and plant development were relatively higher in the early‐successional forest. Seedling establishment success was not associated with specific physiological and morphological adaptations or habitat preference of the studied species. Our results were not consistent with the described community composition and rates of population recruitment of the studied species in the same successional habitats. The results support the hypothesis that chance and historic events related to seed dispersal have an important influence on community assembly of epiphytic plants. In addition, differences in growth rates and reproductive turnover among species are expected to influence the relative abundance and recruitment rates in a particular habitat.
The Effects of Summary Information on Consumer Perceptions of Mutual Fund Characteristics
Authors: KOZUP JOHN , HOWLETT ELIZABETH , PAGANO MICHAEL
Published: February 2008

Choosing how to best invest for retirement is one of the most important decisions a consumer can make. Unfortunately, this can be an especially challenging task given the current financial information disclosure environment. The objective of this research was to explore whether a modified method of supplemental information disclosure impacts investors’ fund evaluations and investment intentions. Results indicate that while investors continue to place too much emphasis on prior performance, the provision of supplemental information, particularly in a graphical format, interacts with performance and investment knowledge to influence perceptions and evaluations of mutual funds.
Use, Misuse, and Abuse of Content Analysis for Research on the Consumer Interest
Authors: CARLSON LES
Published: February 2008

While possessing a long and respected history as a research method, content analysis studies attempting to address issues of the consumers’ interests often are published with invalid conclusions or implications. This essay offers that the source of these problems is rooted in inferring causal relations from what is nothing more than descriptive data.
Novel Molecular Targets for Antimalarial Drug Development
Authors: Sahu Nitendra K. , Sahu Sanjeev , Kohli Dharm Veer
Published: February 2008

Malaria with one million deaths and about 500 million new cases reported annually is a challenge to drug therapy and discovery. As current antimalarial therapeutics become increasingly ineffective because of parasitic resistance, there exists an urgent need to develop and pursue new therapeutic strategies. Antimalarial drug development can follow several strategies, ranging from minor modifications of existing agents to the design of novel agents that act against new targets. Recent advances in our knowledge of parasite biology as well as the availability of the genome sequence provide a wide range of novel targets for drug design. Several promising targets for drug intervention have been revealed in recent years. This review discusses novel molecular targets of the malaria parasite available to the drug discovery scientist.
Changes in the contractile properties of motor units in the rat medial gastrocnemius muscle after one month of treadmill training
Authors: Pogrzebna M. , Celichowski J.
Published: February 2008

Aim: The influence of 4 weeks treadmill training on the contractile properties of motor units (MUs) in the rat medial gastrocnemius muscle was investigated.

Methods: A population of 18 Wistar rats was divided into two groups: trained on a treadmill (n = 7, locomotion speed 27 cm s−1, 1 km daily, 5 days a week, for 4 weeks) and control (n = 11). The contractile properties of isolated MUs were studied. Functional isolation of units was achieved by electrical stimulation of filaments of the ventral roots. A total of 299 MUs were investigated (142 in the control group and 157 in the trained group). They were divided into fast fatigable (FF), fast resistant to fatigue (FR) and slow (S). Their proportions and parameters of contractions were analysed.

Results: Following training, the number of FF units decreased and the number of FR units increased. The distribution of the fatigue index changed within these two types of fast units. The twitch and tetanus forces increased considerably in fast MUs, mainly in those of the FF type. The contraction and relaxation times shortened in the FR and S MUs. The steep part of the force–frequency curves shifted towards higher stimulation frequencies in FR and S units, while in FF units the shift was in the opposite direction.

Conclusion: The significant change in the proportions of fast MUs following training indicates FF to FR transformation. The various effects of training seen in the different MU types help explain the rationale behind mixed training.
Relationships between Ultimate pH and Microbial, Chemical, and Physical Characteristics of Vacuum‐Packaged Pork Loins
Authors: Knox B.L. , Van Laack R.L.J.M. , Davidson P.M.
Published: February 2008

ABSTRACT:  This study evaluated the effect of ultimate pH (pHu) of pork on shelf life based upon microbial growth, drip loss, and oxidative rancidity (2‐thiobarbituric acid [TBA] procedure) in vacuum‐packaged loins stored at 4 °C. Glucose and lactate concentrations of the pork loins were also measured. Thirty‐six pork loins (pH = 5.56 to 6.57) were collected at a commercial slaughter facility 1‐d postslaughter. All pigs were from the same genetic line. Loins were grouped by pH (group: pH range): A: 5.55 to 5.70, B: 5.71 to 5.85, C: 5.86 to 6.00, D: 6.01 to 6.15, and E: > 6.16. They were analyzed at days 0, 6, 14, 24, and 34. For aerobic plate counts, groups A and B were significantly lower than C through E, while psychrotrophic or Enterobacteriaceae counts of groups A and A through C were significantly lower than groups B through E and D and E, respectively. Lactic acid bacteria counts were not significantly influenced by pHu. Group A had higher glucose concentrations than groups C through E and higher lactate concentrations than groups D through E on most sampling days. Group A had a higher TBA value than group E at days 0 and 34. Group A displayed greater drip loss than groups D and E at day 6 and groups B through D on days 24 and 34. Based on the microbial and drip loss results, a pork loin pHu of 5.8 to 5.9 appears to be optimum to provide a vacuum‐packaged shelf life of at least 24 d with minimum drip loss.
Phytosterol Effects on Milk and Yogurt Microflora
Authors: Monu E. , Blank G. , Holley R. , Zawistowski J.
Published: February 2008

ABSTRACT:  Phytosterols play a major role in functional foods. Their ability to reduce serum cholesterol in humans has been widely proven and they are now being added to various dairy based products. The present study investigated the potential antimicrobial activity of phytosterols in milk and their effect on yogurt starter cultures. A commercial phytosterol (0.26% to 1.8%, w/v) preparation (CPP) consisting of ß‐sitosterol, campesterol, sitostanol, and campestanol had no effect on the standard plate count (SPC) and psychrotroph population in pasteurized milk stored at 4 °C. In addition, a challenge study employing Pseudomonas spp. in milk at 4 to 7 °C confirmed that the CPP was not antimicrobial. However, the addition of a dispersible CPP consisting of 0.72% phytosterol containing 0.02% to 0.03% sodium stearoyl lactylate (SSL) did appear to affect the SPC and psychrotrophic bacteria in refrigerated milk. The dispersible preparations did not, however, inhibit the growth of Pseudomonas. An investigation into the antimicrobial activity of SSL revealed that it alone had no effect on the SPC in milk. The CPP had no effect on growth and acid development by Lactobacillus bulgaricus and Streptococcus thermophilus during yogurt production at 33 °C and storage at 4 °C for 30 d. This is seen as a beneficial feature since growth and acid development by these organisms are crucial for yogurt quality. Saccaromyces cerevisiae and Aspergillus ochraceous added to yogurt as typical contaminants also were not inhibited. While the CCP was somewhat antimicrobial when formulated with dispersing agents, it otherwise had no antimicrobial activity.
Myocardial ischaemia–reperfusion injury in haematopoietic cell‐restricted β1 integrin knockout mice
Authors: Metzler Bernhard , Haubner Bernhard , Conci Elisabetta , Voelkl Jakob , Jehle Johannes , Bauer Martina , Wolf Dominik , Pachinger Otmar , Xu Qingbo
Published: February 2008

Evidence indicates that the intercellular adhesion molecule‐1 and its counter‐receptor β2 integrin are cardioprotective proteins during myocardial ischaemia–reperfusion, but no data are available concerning the role of blood cell β1 integrins in this process. We studied the effects of temporary myocardial ischaemia and reperfusion in blood cell‐restricted β1 integrin knockout mice (β1−/−). The left descending coronary artery in conditional β1−/− integrin (β1−/−), β1 integrin +/+ (β1+/+) and β1 integrin −/− bone marrow chimeric (β1−/− BM) mice was ligated for 30 min, followed by reperfusion of either 3 h or 3 weeks. Plasma levels of troponin T were evaluated as an index of cardiac cellular damage. The histological evaluation of tissue damage was performed with Haematoxylin and Eosin stained sections. Cell infiltrations in the ischaemic area were investigated by immunofluorescence studies. It was found that plasma troponin T was at a similar level in β1−/−, β1+/+ and β1−/− BM mice treated with 30 min ischaemia and 3 h reperfusion. Histological analysis showed that ischaemia–reperfusion resulted in marked myocardial injury in all groups of animals, but the damage score of the hearts was not significantly different between β1−/−, β1+/+ and β1−/− BM mice after 3 h of reperfusion following 30 min of ischaemia (2.8 ± 0.5, 2.6 ± 0.5 and 2.8 ± 0.6, respectively, n.s.). Furthermore, no difference in scar sizes in ischaemia‐injured hearts was found 3 weeks after ischaemia. Semi‐quantification of cells demonstrated that, compared with β1+/+ mice, the number of infiltrating neutrophils was significantly reduced in β1−/− and β1−/− BM mice, whereas MAC‐1(CD11b/CD18)‐positive cells in the ischaemic regions were similar in myocardial tissues of all groups. We conclude that absence of β1 integrin expression in haematopoietic cells results in reduced neutrophil infiltration in the ischaemic regions, but does not influence myocardial damage of ischaemic hearts.
Species-Specific Identification of Campylobacters by PCR-Restriction Fragment Length Polymorphism and PCR Targeting of the Gyrase B Gene
Authors: Kawasaki Susumu , Fratamico Pina M. , Wesley Irene V. , Kawamoto Shinichi
Published: February 2008

PCR-restriction fragment length polymorphism (RFLP) analysis of a 960-bp fragment of the
Campylobacter gyrB
gene with either DdeI or XspI restriction enzymes generated unique digestion patterns for 12 different
Campylobacter
species. In addition, PCR assays using species-specific primer sets targeting
gyrB
were specific for the respective
Campylobacter
species. Therefore, PCR-RFLP analysis and species-specific PCR assays based on the
gyrB
gene provide valuable tools for rapid and unambiguous identification of the majority of
Campylobacter
species.
Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide
Authors: Wagner Andreas O. , Malin Cornelia , Knapp Brigitte A. , Illmer Paul
Published: February 2008

Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.
Role of Ureolytic Activity in
Bacillus cereus
Nitrogen Metabolism and Acid Survival
Authors: Mols Maarten , Abee Tjakko
Published: February 2008

The presence and activities of urease genes were investigated in 49 clinical, food, and environmental
Bacillus cereus
isolates. Ten strains were shown to have urease genes, with eight of these strains showing growth on urea as the sole nitrogen source. Two of the urease-positive strains, including the sequenced strain ATCC 10987, could not use urea for growth, despite their capacities to produce active urease. These observations can be explained by the inability of the two strains to use ammonium as a nitrogen source. The impact of urea hydrolysis on acid stress resistance was subsequently assessed among the ureolytic
B. cereus
strains. However, none of the strains displayed increased fitness under acidic conditions or showed enhanced acid shock survival in the presence of urea. Expression analysis of urease genes in
B. cereus
ATCC 10987 revealed a low level of expression of these genes and a lack of pH-, nitrogen-, urea-, oxygen-, and growth phase-dependent modulation of mRNA transcription. This is in agreement with the low urease activity observed in strain ATCC 10987 and the other nine strains tested. Although a role for
B. cereus
ureolytic activity in acid survival cannot be excluded, its main role appears to be in nitrogen metabolism, where ammonium may be provided to the cells in nitrogen-limited, urea-containing environments.
Promoter Activation by Repositioning of RNA Polymerase
Authors: Kumar Amrita , Moran Charles P.
Published: February 2008

Spo0A, a classical two-component-type response regulator in
Bacillus subtilis
, binds to a specific DNA sequence found in many promoters to repress or activate the transcription of over 100 genes. On the
spoIIG
promoter, one of the Spo0A binding sites, centered at position −40, overlaps a consensus −35 element that may also interact with region 4 of the sigma A (σ
A
) subunit of RNA polymerase. Molecular modeling corroborated by genetic evidence led us to propose that the binding of Spo0A to this site repositions σ
A
region 4 on the promoter. Therefore, we used a chemical nuclease,
p
-bromoacetamidobenzyl-EDTA-Fe, that was covalently tethered to a single cysteine in region 4 of σ
A
to map the position of σ
A
on the promoter. The results indicated that in the absence of Spo0A, σ
A
region 4 of the RNA polymerase was located near the −35 element sequence centered at position −40. However, in the presence of Spo0A, σ
A
region 4 was displaced downstream from the −35 element by 4 bp. These and other results support the model in which the binding of Spo0A to the
spoIIG
promoter stimulates promoter utilization by repositioning prebound RNA polymerase and stabilizing the repositioned RNA polymerase-promoter complex at a new position that aligns σ
A
region 2 with the −10 region sequences of the promoter, thus facilitating open complex formation.
Two RecA Protein Types That Mediate Different Modes of Hyperrecombination
Authors: Baitin Dmitry M. , Bakhlanova Irina V. , Chervyakova Darya V. , Kil Yury V. , Lanzov Vladislav A. , Cox Michael M.
Published: February 2008

RecAX53 is a chimeric variant of the
Escherichia coli
RecA protein (RecAEc) that contains a part of the central domain of
Pseudomonas aeruginosa
RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in
E. coli
cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the
mutS
gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers.
RelA Functionally Suppresses the Growth Defect Caused by a Mutation in the G Domain of the Essential Der Protein
Authors: Hwang Jihwan , Inouye Masayori
Published: February 2008

A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit inEscherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of anE. coligenomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on theE. coligenomic map, and the gene responsible for the suppression of DerN321D was identified as therelAgene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.
ArnR, a Novel Transcriptional Regulator, Represses Expression of the
narKGHJI
Operon in
Corynebacterium glutamicum
Authors: Nishimura Taku , Teramoto Haruhiko , Vertès Alain A. , Inui Masayuki , Yukawa Hideaki
Published: February 2008

The
narKGHJI
operon that comprises putative nitrate/nitrite transporter (
narK
) and nitrate reductase (
narGHJI
) genes is required for the anaerobic growth of
Corynebacterium glutamicum
with nitrate as a terminal electron acceptor. In this study, we identified a gene,
arnR
, which encodes a transcriptional regulator that represses the expression of the
narKGHJI
operon in
C. glutamicum
cells under aerobic conditions. Disruption of
arnR
induced nitrate reductase activities of
C. glutamicum
cells and increased
narKGHJI
mRNA levels under aerobic growth conditions. DNA microarray analyses revealed that besides the
narKGHJI
operon, the
hmp
gene, which encodes flavohemoglobin, is negatively regulated by ArnR under aerobic conditions. Promoter-reporter assays indicated that
arnR
gene expression was positively autoregulated by its gene product, ArnR, under both aerobic and anaerobic conditions. Electrophoretic mobility shift assay experiments showed that purified hexahistidyl-tagged ArnR protein specifically binds to promoter regions of the
narKGHJI
operon and the
hmp
and
arnR
genes. A consensus sequence, TA(A/T)TTAA(A/T)TA, found in the promoter regions of these genes was demonstrated to be involved in the binding of ArnR. Effects on LacZ activity by deletion of the ArnR binding sites within the promoter regions fused to the reporter gene were consistent with the view that the expression of the
narKGHJI
operon is repressed by the ArnR protein under aerobic conditions, whereas the expression of the
arnR
gene is autoinduced by ArnR.
Microcompartments for B
12
-Dependent 1,2-Propanediol Degradation Provide Protection from DNA and Cellular Damage by a Reactive Metabolic Intermediate
Authors: Sampson Edith M. , Bobik Thomas A.
Published: February 2008

Salmonella enterica
grows on 1,2-propanediol (1,2-PD) in a coenzyme B
12
-dependent fashion. Prior studies showed that a bacterial microcompartment (MCP) is involved in this process and that an MCP-minus mutant undergoes a 20-h period of growth arrest during 1,2-PD degradation. It was previously proposed that growth arrest resulted from propionaldehyde toxicity, but no direct evidence was presented. Here, high-pressure liquid chromatography analyses of culture medium were used to show that the major products of aerobic 1,2-PD degradation are propionaldehyde, propionate, and 1-propanol. A MCP-minus mutant accumulated a level of propionaldehyde 10-fold higher than that of the wild type (1.6 mM compared to 15.7 mM), associating this compound with growth arrest. The addition of propionaldehyde to cultures of
S. enterica
caused growth arrest from 8 to 20 mM, but not at 4 mM, providing direct evidence for propionaldehyde toxicity. Studies also indicated that propionaldehyde was toxic due to the inhibition of respiratory processes, and the growth arrest ended when propionaldehyde was depleted primarily by conversion to propionate and 1-propanol and secondarily due to volatility. The Ames test was used to show that propionaldehyde is a mutagen and that mutation frequencies are increased in MCP-minus mutants during 1,2-PD degradation. We propose that a primary function of the MCPs involved in 1,2-PD degradation is the mitigation of toxicity and DNA damage by propionaldehyde.
DivIVA Is Required for Polar Growth in the MreB-Lacking Rod-Shaped ActinomyceteCorynebacterium glutamicum
Authors: Letek Michal , Ordóñez Efrén , Vaquera José , Margolin William , Flärdh Klas , Mateos Luis M. , Gil José A.
Published: February 2008

The actinomyceteCorynebacterium glutamicumgrows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVACg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA fromStreptomycesorMycobacteriumlocalized to the cell poles of DivIVACg-depletedC. glutamicumand restored polar peptidoglycan synthesis, in contrast to DivIVA proteins fromBacillus subtilisorStreptococcus pneumoniae, which localized at the septum ofC. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVACglocalized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that inC. glutamicumDivIVA is not involved in cell division or chromosome segregation.
Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression
Authors: Kabaeva Zhyldyz , Zhao Mei , Michele Daniel E.
Published: February 2008

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.
RNAi inhibition of mineralocorticoid receptors prevents the development of cold-induced hypertension
Authors: Sun Zhongjie , Bello-Roufai Mahajoub , Wang Xiuqing
Published: February 2008

The objective was to determine whether the mineralocorticoid receptor (MR) plays a role in the initiation and development of cold-induced hypertension (CIH) by testing the hypothesis that the RNA interference (RNAi) inhibition of the MR attenuates CIH. The recombinant adeno-associated virus (AAV) carrying a short-hairpin small-interference RNA for MR (MRshRNA) or a scrambled sequence (ControlshRNA) was constructed. Six groups of albino mice were used (6 mice/group). Three groups were exposed to cold (6.7°C), whereas the remaining three groups were kept at room temperature (RT; warm) as controls. In each temperature condition, three groups received an intravenous injection of MRshRNA, ControlshRNA, or virus-free PBS, respectively, before exposure to cold. The viral complexes (0.35 × 1011 particles/mouse, 0.5 ml) or PBS (0.5 ml) was delivered into the circulation via the tail vein. The blood pressure (BP) of the mice treated with ControlshRNA or PBS increased significantly during exposure to cold, whereas the BP of the cold-exposed MRshRNA-treated mice did not increase and remained at the level of the control group kept at RT. Thus AAV delivery of MRshRNA prevented the initiation of CIH. MRshRNA significantly attenuated cardiac and renal hypertrophy. MRshRNA decreased the cold-induced increase in MR protein expression to the control level in the hypothalamus, kidneys, and heart, indicating an effective prevention of the cold-induced upregulation of MR. RNAi inhibition of MR resulted in significant decreases in the plasma level of norepinephrine, plasma renin activity, and plasma level of aldosterone in cold-exposed mice. MR played a critical role in the initiation and development of CIH. AAV delivery of MRshRNA may serve as a new approach for the prevention of cold-induced hypertension.
Identification of Flotillin-2, a Major Protein on Lipid Rafts, as a Novel Target of p53 Family Members
Authors: Sasaki Yasushi , Oshima Yuichiro , Koyama Ryota , Maruyama Reo , Akashi Hirofumi , Mita Hiroaki , Toyota Minoru , Shinomura Yasuhisa , Imai Kohzoh , Tokino Takashi
Published: February 2008

p73 and p63 are members of the p53 gene family and have been shown to play an important role in development and homeostasis mainly by regulating the transcription of a variety of genes. A subset of these genes encodes secreted proteins and receptors that may be involved in the communication between adjacent cells. We report here that flotillin-2, a major hydrophobic protein on biomembrane microdomain lipid rafts, is a direct transcriptional target of the p53 family member genes. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. We found that the expression of flotillin-2 was specifically up-regulated by either TAp73β or TAp63γ, but not significantly by p53. In addition, flotillin-2 transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we showed that silencing endogenous p73 abolishes the induction of flotillin-2 transcription following cisplatin treatment. Furthermore, we identified a p73/p63-binding site located upstream of the flotillin-2 gene that is responsive to the p53 family members. This response element is highly conserved between humans and rodents. We also found that ectopic expression of TAp73 as well as TAp63 enhances signal transduction by assessing the interleukin-6–mediated phosphorylation of signal transducers and activators of transcription 3. Thus, in addition to direct transactivation, p53 family member genes enhance a set of cellular processes via lipid rafts. (Mol Cancer Res 2008;6(3):395–406)
Binding of the Retinoblastoma Protein Is Not the Determinant for Stable Repression of Some E2F-Regulated Promoters in Muscle Cells
Authors: Vandromme Marie , Chailleux Catherine , Escaffit Fabrice , Trouche Didier
Published: February 2008

Permanent silencing of E2F-dependent genes is a hallmark of the irreversible cell cycle exit that characterizes terminally differentiated and senescent cells. The determinant of this silencing during senescence has been proposed to be the binding of the retinoblastoma protein Rb and the consequent methylation of H3K9. During ex vivo skeletal muscle differentiation, while most cells terminally differentiate and form myotubes, a subset of myoblasts remains quiescent and can be reinduced by growth factor stimulation to enter the cell cycle. Thus, differentiating cells are composed of two different populations: one in which E2F-dependent genes are permanently repressed and the other not. We observed that, in a manner reminiscent to senescent cells, permanent silencing of the E2F-dependent cdc6, dhfr, and p107 promoters in myotubes was associated with a specific increase in H3K9 trimethylation. To investigate the role of Rb in this process, we developed a reliable method to detect Rb recruitment by chromatinimmunoprecipitation. Surprisingly, we observed that Rb was recruited to these promoters more efficiently in quiescent cells than in myotubes. Thus, our data indicate that during muscle differentiation, permanent silencing and H3K9 trimethylation of some E2F-dependent genes are not directly specified by Rb binding, in contrast to what is proposed for senescence. (Mol Cancer Res 2008;6(3):418–25)
Myocardial Pitx2 Differentially Regulates the Left Atrial Identity and Ventricular Asymmetric Remodeling Programs
Authors: Tessari Alessandra , Pietrobon Mara , Notte Antonella , Cifelli Giuseppe , Gage Philip J. , Schneider Michael D. , Lembo Giuseppe , Campione Marina
Published: February 2008

The Pitx2 gene regulates left-right (L/R) asymmetrical cardiac morphogenesis. Constitutive Pitx2 knock out (ko) mice die before birth and display, among other defects, right atrial isomerism, atrial and ventricular septal defects, and double outlet right ventricle. The myocardial role of the gene has not been dissected. In particular, how Pitx2 regulates the differential L/R cardiac identity program is not clear. Additionally, the relation between Pitx2 ko ventricular defects and the gene expression pattern is not understood. In this article we analyze Pitx2 myocardial function during mouse heart development. By in situ hybridization analysis we show that myocardial Pitx2 expression delineates the remodeling of the left atrioventricular canal, the inner curvature, the ventral part of the interventricular ring, and the ventral portion of the right and left ventricle. By genetic analysis using an allelic series of Pitx2 mutants, among which a myocardial specific ko (ko
myo
) we show it has a crucial role in this process. Pitx2 ko
myo
mutants survive to adulthood, when they present strong cardiac morphological and functional defects. Confocal analysis of embryonic Pitx2 ko
myo
hearts reveals delayed cardiomyocyte development in the ventricular but not in the atrial Pitx2 null areas. Conversely, selective left atrial BMP10 mRNA downregulation which normally occurs at fetal stages is not found in the Pitx2 ko
myo
mice. This is the first evidence for distinct Pitx2 action in mediating L/R atrial identity and asymmetrical ventricular remodeling.