bluuebunny/crossref_metadata_2025 huggingface dataset:
T lymphocyte responses of sheep to bovine leukaemia virus infection
Authors: WARD W. H. , DIMMOCK C. K. , EAVES F. W.
Published: July 2007

Sheep were experimentally infected with bovine leukaemia virus (BLV) by inoculation of peripheral blood lymphocytes (PBL) from BLV infected sheep. Monoclonal antibodies were used to monitor changes in lymphocyte subpopulations in the first few weeks after inoculation. The polymerase chain reaction (PCR) detected BLV DNA in PBL of infected sheep 11‐15 days after inoculation, that is, before antibodies to viral structural proteins were detected at 15‐39 days post‐inoculation. A rise in the number of both B and T lymphocytes coincided with detection of infection by PCR. At this time, an increase in the number of circulation CD8+ lymphocytes resulted in a low CD4: CD8 ratio. It appears that in BLV infection there is a host specific cell‐mediated immune response to infected lymphocytes rather than a general immune response to foreign antigens. This response, which is characterized by an increase in the number of circulating CD8+ lymphocytes, precedes seroconversion. There is considerable variation between animals in this cytotoxic T lymphocyte response.
Inhibition of adjuvant arthritis in the rat by phosphosugars and the α‐glucosidase inhibitor castanospermine
Authors: WILLENBORG DAVID O. , PARISH CHRISTOPHER R. , COWDEN WILLIAM B.
Published: July 2007

The development of joint inflammation of adoptively transferred arthritis in rats was inhibited by treatment with the simple sugar mannose‐6‐phosphate or the alkaloid inhibitor of α‐glucosidase, castanospermine. Mannose‐6‐phosphate was effective at a dose of 25 mg/kg per day delivered via mini‐osmotic pumps implanted either subcutaneously or intraperitoncally. Castanospermine was given orally in the drinking water and rats ingested on average 60‐65 mg/kg per day. Histological examination of tissue from treated rats revealed greatly reduced inflammatory infiltrates into the synovium and surrounding tissue. Castanospermine not only inhibited the development of arthritis but also inhibited the progression of the disease when treatment was commenced after the onset of symptoms. Possible mechanism(s) of action of these compounds could be their ability to inhibit the passage of leucocytes through vascular subendothelial basement membranes by inhibiting the function or expression of leucocyte cell surface‐bound enzymes that are essential for such migration. Castanospermine could also inhibit inflammation through its ability to prevent the expression of adhesion molecules, which may be necessary for the capture and retention of leucocytes in the inflamed tissue.
Generation and cytotoxic profile of human peripheral blood CD4 + T lymphocytes
Authors: SMYTH MARK J.
Published: July 2007

The effects of a variety of metabolic and anti‐tumour necrosis factor (TNF) antibodies were utilized to distinguish several different mechanisms of cytotoxicity employed by CD4+ effectors isolated from human peripheral blood lymphocytes (PBL). PBL, unseparated high buoyant density T cells and their CD4+ T cell subsets were activated with anti‐CD3 monoclonal antibody (MoAb) and interleukin‐2 (IL‐2) for 1‐5 days. CD4+ T cells activated with IL‐2/anti‐CD3 MoAb were cytotoxic when directed by a bispecific anti‐nitrophenyl (NP)‐anti‐CD3 MoAb heteroconjugate against both NP‐modified nucleated target cells (TC) and non‐nucleated sheep red blood cells (SPLBC). This CD4+ T population also lysed L929 in a TNF‐a dependent manner. Interestingly, different mechanisms of nucleated and non‐nucleated TC directed lysis by CD4+ effectors were implied by distinct patterns of sensitivity to cholera toxin (CT) and cyclosporin A (CsA). Cyclosporin A and CT inhibited CD4+ T cell directed lysis of SRBC, but not EL4. Cholera toxin, CsA or EGTA pretreatment also significantly inhibited the release of α‐N‐benzyloxycarbonyl‐l‐lysinethiobenzylester (BLT)‐esterase activity suggesting that degranulation of CD4+ effectors may be a critical step in their redirected lysis of SRBC. Overall, these findings suggested that activated human peripheral blood (PB) CD4+ effectors can lyse TC by at least three distinct mechanisms: (i) a CsA‐sensitive directed lysis of SRBC which correlates with exocytosis and presumably occurs via membrane lesions; (ii) a CsA‐insensitive directed lysis of NP‐modified nucleated TC that does not appear to involve exocytosis and is metabolically distinct; and (iii) a direct TNF‐dependent lysis of TNF‐sensitive TC. The highly prolifetative CD4+ T cell population could be propagated for at least 35 days while retaining cytotoxicity and secreting up to 80 U/mL of IL‐2. These data raise the possibility that anti‐CD3 MoAb plus IL‐2 activated CD4+ T cells may prove effective in adoptive tumour immunotherapy.
Development of human ABO blood group A antigen on Escherichia coli Y1089 and Y1090
Authors: YANG NAN , BOETTCHER BARRY
Published: July 2007

Studies by other workers have shown that some strains of Escherichia coli have surface antigens analogous to the human blood group ABH antigens, and that these are carbohydrates associated with membrane lipopolysaccharides. This study has demonstrated that E. coli strains Y1089 and Y1090 possess the H antigen, which can be converted to the A antigen by incubation with A‐transferase (N‐acetyl‐galactosaminyl transferase) and A‐sugar (UDP‐N‐acetyl‐galactosamine). Such cells will then form mixed agglutinates with human A red cells and human polyclonal (but not mouse monoclonal) anti‐A antibodies. E. coli Y1089 and Y1090 have endogenous enzymes that use the A‐sugar (in the absence of A‐ transferase) to produce a variant A antigen. Cells expressing this variant antigen adsorb anti‐A antibodies but do not participate in mixed agglutination with human group A red cells. It is estimated that E. coli Y1089 and Y1090 possess approximately 5000 H epitopes per cell that can be converted to A epitopes.
Enhanced H2O2 release from immune chicken leucocytes following infection with Eimeria tenella
Authors: PROWSE S.J. , MICHALSKI W. P. , FAHEY K. J.
Published: July 2007

Cell‐mediated immunity is thought to be important in the resistance of chickens to infection by coccidia, and it has been demonstrated that sporozoites of Eimeria tenella are very sensitive to superoxide ions. Therefore an investigation into the cellular responses in naive specific pathogenfree and hyperimmune birds was carried out with particular attention to their ability to produce reactive derivatives of oxygen. Leucocytes were isolated from the blood, spleen and caecal mucosa of chickens infected with E. tenella and assessed for their ability to release H2O2‐ Leucocytes obtained from the blood and spleen of hyperimmune birds 1 day after challenge showed an elevated ability to produce reactive oxygen intermediates. In contrast, the ability of leucocytes from naive chickens to produce these molecules was transiently depressed after challenge. Prior to challenge, mucosal leucocytes from immune chickens were also able to release heightened levels of H2O2 when compared with cells from naive chickens.
Viral escape from immune recognition: Multiple strategies of adenoviruses
Authors: MÜLLBACHER ARNO
Published: July 2007

Human adenovirases can cause persistent infections in man. The strategies of C type adenoviruses (types Ad2 and Ad5) to evade immune recognition are many, but all involve single early genetic regions (E3). Gene(s) within E3 have been shown to allow the adenovirus to avert cytokine‐mediated apoptosis. Furthermore, the E3 region controls the cytotoxic T cell epitope (a major histocompatibility complex [MHC] class I molecule with an adenovirus‐derived peptide) on the cell surface of infected cells. On the one hand the E3 gene product 19 kDa can bind to nascent class I MHC in the endoplasmic reticulum and thus prevent its transport to the cell surface, and on the other hand the E3 region down‐regulates the Ela gene product, the immunodominant cytotoxic T cell determinant.
Immortalized Human Brain Endothelial Cells and Flow-Based Vascular Modeling: A Marriage of Convenience for Rational Neurovascular Studies
Authors: Cucullo Luca , Couraud Pierre-Olivier , Weksler Babette , Romero Ignacio-Andres , Hossain Mohammed , Rapp Edward , Janigro Damir
Published: July 2007

In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood—Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope = 0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1β) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.
Diurnal expression of five protein phosphatase type 2C genes in the common ice plant, Mesembryanthemum crystallinum
Authors: Sato Ko , Ohsato Hiroaki , Izumi Shunsuke , Miyazaki Saori , Bohnert Hans J. , Moriyama Hiromitsu , Fukuhara Toshiyuki
Published: July 2007

The common ice plant, Mesembryanthemum crystallinum L., is a eu-halophytic model species with an environmental stress-initiated switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Phosphoenolpyruvate carboxylase activity in 6-week-old plants exposed to salt stress for 5 days was ~15-fold higher than before stress, indicating the salinity-dependent induction of the C3 to CAM transition. Five plant protein phosphatase type 2C (PP2C) genes were cloned, representative of five of the 10 plant PP2C sub-families. We measured mRNA levels of these PP2Cs and of myo-inositol 1-phosphate synthase (Inps1) in 6-week-old plants before (C3) and after (CAM) salt stress. Remarkably, four PP2C genes and Inps1 were expressed with a diurnal fluctuation in plants in C3 mode. After salt-induced CAM induction, the six genes were expressed with more prominent fluctuations than before stress, suggesting that these PP2C genes may be involved in the diurnal regulation of protein phosphorylation in CAM. Under continuous light treatment the expression of two PP2C genes continued to fluctuate, indicating that their expression is controlled by circadian rhythm.
Expression of a human tRNA isopentenyltransferase in tobacco reveals a developmental role for tRNA isopentenyladenosine
Authors: Golovko Anna , Sitbon Folke , Tillberg Elisabeth , Nicander Björn
Published: July 2007

In addition to their role as plant hormones, cytokinins are also found as structural components in tRNA. Six different tRNA cytokinins have been found in plants, but most other organisms, including humans, have only one–isopentenyladenosine. In an attempt to probe if the different forms have different functionality, we attempted to alter tRNA cytokinin composition by expressing the human tRNA isopentenyltransferase gene (EC 5.1.2.8) in tobacco [Nicotiana tabacum (L.) cv. Wisconsin 38]. The resulting transgenics had ~40% more isopentenyladenosine in tRNA, and an altered phenotype characterised by reduced internode length, increased stem diameter and rigidity, greener leaves, increased axillary bud outgrowth, abnormal flower morphology, and reduced seed viability. The levels of the two other major isoprene adenines of tRNA, cis-zeatin and 2-methyltiolated cis-zeatin, were also increased, but to a lower degree. Nearly all of the increase in isopentenyladenosine was in a single tRNA species. Two quantitatively minor isopentenyladenosine-containing tRNAs had also increased strongly. IPPT: Dimethylallylpyrophosphate:
A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole
Authors: Jackson Mark A. , Rae Anne L. , Casu Rosanne E. , Grof Christopher P. L. , Bonnett Graham D. , Maclean Donald J.
Published: July 2007

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products.
TNF-induced structural joint damage is mediated by IL-1
Authors: Zwerina Jochen , Redlich Kurt , Polzer Karin , Joosten Leo , Krönke Gerhard , Distler Joerg , Hess Andreas , Pundt Noreen , Pap Thomas , Hoffmann Oskar , Gasser Juerg , Scheinecker Clemens , Smolen Josef S. , van den Berg Wim , Schett Georg
Published: July 2007

Blocking TNF effectively inhibits inflammation and structural damage in human rheumatoid arthritis (RA). However, so far it is unclear whether the effect of TNF is a direct one or indirect on up-regulation of other mediators. IL-1 may be one of these candidates because it has a central role in animal models of arthritis, and inhibition of IL-1 is used as a therapy of human RA. We removed the effects of IL-1 from a TNF-mediated inflammatory joint disease by crossing IL-1α and β-deficient mice (IL-1−/−) with arthritic human TNF-transgenic (hTNFtg) mice. Development of synovial inflammation was almost unaffected on IL-1 deficiency, but bone erosion and osteoclast formation were significantly reduced in IL-1−/−hTNFtg mice, compared with hTNFtg mice based on an intrinsic differentiation defect of IL-1-deficient monocytes. Most dramatically, however, cartilage damage was absent in IL-1−/−hTNFtg mice. Chimera studies revealed that protection of cartilage is based on the loss of IL-1 on hematopoietic, but not mesenchymal, cells, leading to decreased expression of ADAMTS-5 and MMP-3. These data show that TNF-mediated cartilage damage is completely and TNF-mediated bone damage is partially dependent on IL-1, suggesting that IL-1 is a crucial mediator for inflammatory cartilage and bone degradation.
Telomerase core components protect
Candida
telomeres from aberrant overhang accumulation
Authors: Hsu Min , McEachern Michael J. , Dandjinou Alain T. , Tzfati Yehuda , Orr Erica , Blackburn Elizabeth H. , Lue Neal F.
Published: July 2007

Telomerase is a cellular reverse transcriptase that extends one strand (the G-strand) of the telomere terminal repeats. Aside from this role in telomere length maintenance, telomerase has been proposed to serve a protective function at chromosome ends, although this is not well understood mechanistically. Earlier analysis suggests that, in the pathogenic yeast
Candida albicans
, the catalytic reverse transcriptase subunit of telomerase (TERT/EST2) can protect telomeres against nucleolytic degradation. In this report we demonstrate that the RNA component (TER1) has a similar function; in addition to complete loss of telomerase activity and progressive telomere attrition, the
ter1
-ΔΔ strains manifested a dramatic increase in the amount of G-strand overhangs, consistent with aberrant degradation of the complementary C-strand. We also demonstrate that a catalytically incompetent EST2 protein can suppress such overhang accumulation in the
est2
-ΔΔ mutant to the same extent as the wild-type protein. Altogether, our data support the notion that the
Candida
telomerase core components mediate a protective function through a mechanism that is independent of its catalytic activity.
Mycobacterium tuberculosis
WhiB3 responds to O
2
and nitric oxide via its [4Fe-4S] cluster and is essential for nutrient starvation survival
Authors: Singh Amit , Guidry Loni , Narasimhulu K. V. , Mai Deborah , Trombley John , Redding Kevin E. , Giles Gregory I. , Lancaster Jack R. , Steyn Adrie J. C.
Published: July 2007

A fundamental challenge in the redox biology of
Mycobacterium tuberculosis
(
Mtb
) is to understand the mechanisms involved in sensing redox signals such as oxygen (O
2
), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that
Mtb
WhiB3 responds to the dormancy signals NO and O
2
through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the
Mtb
cysteine desulfurase (IscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in
Mtb
. EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [4Fe-4S]
2+
to EPR visible [4Fe-4S]
+
. Atmospheric O
2
gradually degrades the WhiB3 [4Fe-4S]
2+
cluster to generate a [3Fe-4S]
+
intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl–iron–dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly,
Mtb
Δ
whiB3
shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and O
2
through its [4Fe-4S] cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism.
The design and characterization of two proteins with 88% sequence identity but different structure and function
Authors: Alexander Patrick A. , He Yanan , Chen Yihong , Orban John , Bryan Philip N.
Published: July 2007

To identify a simplified code for conformational switching, we have redesigned two natural proteins to have 88% sequence identity but different tertiary structures: a 3-α helix fold and an α/β fold. We describe the design of these homologous heteromorphic proteins, their structural properties as determined by NMR, their conformational stabilities, and their affinities for their respective ligands: IgG and serum albumin. Each of these proteins is completely folded at 25°C, is monomeric, and retains the native binding activity. The complete binding epitope for both ligands is encoded within each of the proteins. The IgG-binding epitope is functional only in the α/β fold, and the albumin-binding epitope is functional only in the 3-α fold. These results demonstrate that two monomeric folds and two different functions can be encoded with only 12% of the amino acids in a protein (7 of 56). The fact that 49 aa in these proteins are compatible with both folds shows that the essential information determining a fold can be highly concentrated in a few amino acids and that a very limited subset of interactions in the protein can tip the balance from one monomer fold to another. This delicate balance helps explain why protein structure prediction is so challenging. Furthermore, because a few mutations can result in both new conformation and new function, the evolution of new folds driven by natural selection for alternative functions may be much more probable than previously recognized.
Identifying important structural characteristics of arsenic resistance proteins by using designed three-stranded coiled coils
Authors: Touw Debra S. , Nordman Christer E. , Stuckey Jeanne A. , Pecoraro Vincent L.
Published: July 2007

Arsenic, a contaminant of water supplies worldwide, is one of the most toxic inorganic ions. Despite arsenic's health impact, there is relatively little structural detail known about its interactions with proteins. Bacteria such as
Escherichia coli
have evolved arsenic resistance using the Ars operon that is regulated by ArsR, a repressor protein that dissociates from DNA when As(III) binds. This protein undergoes a critical conformational change upon binding As(III) with three cysteine residues. Unfortunately, structures of ArsR with or without As(III) have not been reported. Alternatively,
de novo
designed peptides can bind As(III) in an endo configuration within a thiolate-rich environment consistent with that proposed for both ArsR and ArsD. We report the structure of the As(III) complex of Coil Ser L9C to a 1.8-Å resolution, providing x-ray characterization of As(III) in a Tris thiolate protein environment and allowing a structural basis by which to understand arsenated ArsR.
An NQO1- and PARP-1-mediated cell death pathway induced in non-small-cell lung cancer cells by β-lapachone
Authors: Bey Erik A. , Bentle Melissa S. , Reinicke Kathryn E. , Dong Ying , Yang Chin-Rang , Girard Luc , Minna John D. , Bornmann William G. , Gao Jinming , Boothman David A.
Published: July 2007

Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-small-cell lung cancers (NSCLCs) have a survival rate of ≈15% after 5 years. Novel approaches are needed to treat this disease. We show elevated NAD(P)H:quinone oxidoreductase-1 (NQO1) levels in tumors from NSCLC patients. β-Lapachone, an effective chemotherapeutic and radiosensitizing agent, selectively killed NSCLC cells that expressed high levels of NQO1. Isogenic H596 NSCLC cells that lacked or expressed NQO1 along with A549 NSCLC cells treated with or without dicoumarol, were used to elucidate the mechanism of action and optimal therapeutic window of β-lapachone. NSCLC cells were killed in an NQO1-dependent manner by β-lapachone (LD
50
, ≈4 μM) with a minimum 2-h exposure. Kinetically, β-lapachone-induced cell death was characterized by the following: (
i
) dramatic reactive oxygen species (ROS) formation, eliciting extensive DNA damage; (
ii
) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP-1); (
iii
) depletion of NAD
+
/ATP levels; and (
iv
) proteolytic cleavage of p53/PARP-1, indicating μ-calpain activation and apoptosis. β-Lapachone-induced PARP-1 hyperactivation, nucleotide depletion, and apoptosis were blocked by 3-aminobenzamide, a PARP-1 inhibitor, and 1,2-bis(2-aminophenoxy)ethane-
N
,
N
,
N′
,
N′
-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a Ca
2+
chelator. NQO1
−
cells (H596, IMR-90) or dicoumarol-exposed NQO1
+
A549 cells were resistant (LD
50
, >40 μM) to ROS formation and all cytotoxic effects of β-lapachone. Our data indicate that the most efficacious strategy using β-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1
−
“normal” cells. β-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1
+
NSCLC cancers.
Extracellular structure of polysialic acid explored by on cell solution NMR
Authors: Azurmendi Hugo F. , Vionnet Justine , Wrightson Lauren , Trinh Loc B. , Shiloach Joseph , Freedberg Darón I.
Published: July 2007

The capsular polysaccharide of the pathogens
Neisseria meningitidis
serogroup B and of
Escherichia coli
K1, α(2 → 8) polysialic acid (PSA), is unusual, because when injected into adult humans, it generates little or no antibody. In contrast, people infected with these pathogens generate specific serum antibodies. A structural study on cells is used to address this anomaly by characterizing antigen structures
in vivo
. We introduce on cell multidimensional solution NMR spectroscopy for direct observation of PSA on
E. coli
bacteria. Using
13
C,
15
N-labeled PSA, we applied a combination of heteronuclear NMR methods, such as heteronuclear single quantum coherence, HNCA, and HNCO,
in vivo
. Analysis reveals that free and cell-bound PSA are structurally similar, indicating that the poor immunogenicity of PSA is not due to major structural differences between cells and purified PSA. The
13
C linewidths of PSA on cells are 2 to 3 times larger than the corresponding ones in free PSA. The possible implications of the differences between free and on cell PSA are discussed. In addition, we demonstrate the suitability of the method for
in vivo
kinetic studies.
Laboratory evolution of one disulfide isomerase to resemble another
Authors: Hiniker Annie , Ren Guoping , Heras Begoña , Zheng Ying , Laurinec Stephanie , Jobson Richard W. , Stuckey Jeanne A. , Martin Jennifer L. , Bardwell James C. A.
Published: July 2007

It is often difficult to determine which of the sequence and structural differences between divergent members of multigene families are functionally important. Here we use a laboratory evolution approach to determine functionally important structural differences between two distantly related disulfide isomerases, DsbC and DsbG from
Escherichia coli
. Surprisingly, we found single amino acid substitutions in DsbG that were able to complement
dsbC in vivo
and have more DsbC-like isomerase activity
in vitro
. Crystal structures of the three strongest point mutants, DsbG K113E, DsbG V216M, and DsbG T200M, reveal changes in highly surface-exposed regions that cause DsbG to more closely resemble the distantly related DsbC. In this case, laboratory evolution appears to have taken a direct route to allow one protein family member to complement another, with single substitutions apparently bypassing much of the need for multiple changes that took place over ≈0.5 billion years of evolution. Our findings suggest that, for these two proteins at least, regions important in determining functional differences may represent only a tiny fraction of the overall protein structure.
Akt1 and Akt2 are required for αβ thymocyte survival and differentiation
Authors: Juntilla Marisa M. , Wofford Jessica A. , Birnbaum Morris J. , Rathmell Jeffrey C. , Koretzky Gary A.
Published: July 2007

The β-selection checkpoint in αβT lymphocyte development occurs at the double negative (DN) 3 (CD4−CD8−CD25+c-kit−) stage, when further differentiation requires a signal from the newly rearranged TCR β chain. Thymocytes with mutations in key signaling molecules in the phosphatidylinositol 3-kinase–Akt pathway manifest defects in survival, proliferation, and differentiation past the β-selection checkpoint. However, little information is available regarding the role of Akt itself in thymocyte development. In this study, we explore the role of the two Akt isoforms most highly expressed in the thymus, Akt1 and Akt2, in early T cell development. Using several complementary approaches, we find that deletion of Akt1 results in only minor defects in thymocyte development. The Akt1−/−Akt2−/−thymocytes manifest a severe developmental block at the DN3 stage and ultimately fail to repopulate the T cell compartment of an irradiated host. Further, we show that Akt1−/−Akt2−/−DN3 cells have decreased glucose uptake and die in response to TCR stimulationin vitro. Study of thymocytes from the genetically altered mice suggests that the cause of the developmental defect is due to apoptosis, partially caused by decreased cellular growth and metabolism at the DN3 stage. Our results show that Akt protects thymocytes from cell death during the β-selection checkpoint.
Addiction sciences and its psychometrics: the measurement of alcohol‐related problems
Authors: Midanik Lorraine T. , Greenfield Thomas K. , Bond Jason
Published: July 2007

Aims  The focus of this paper is on psychometric issues related to the measurement of alcohol problems.

Methods  Taking a broad perspective, this paper first examines several issues around the use of instruments to provide diagnostic categories in surveys, including dimensionality, severity and alcohol consumption. Secondly, a discussion of some of the political issues surrounding measurement of alcohol problems is presented, including some of the conflicts that arise when the psychometric properties of commonly used instruments are questioned. Finally, newer statistical techniques that can be applied to scale development in the alcohol field are examined, including non‐linear multivariate analyses and confirmatory/hypothesis‐based methods.

Results and conclusions  Continued scholarly discussion needs to be encouraged around these psychometric issues so that instrument development and maintenance in the addiction sciences becomes an ongoing academic pursuit as we strive to measure alcohol problems in the best way possible.
Primary care prescribing patterns in Ireland after the publication of large hypertension trials
Authors: Kabir Zubair , Feely John , Bennett Kathleen
Published: July 2007

What is already known about this subject

• Between 2000 and 2004, three large hypertension trials were particularly important in choice of antihypertensive drug; LIFE, ALLHAT and VALUE.

• Studies in the US and Canada have shown that subsequent prescribing patterns have been influenced through such publications.

What this study adds

• There was little or no effect of any of the trials on new antihypertensive prescribing in Ireland, except for a slight increase in new prescriptions for ACE inhibitors following ALLHAT, and calcium channel blockers following the VALUE trial.

Aims

This study assessed prescribing patterns of antihypertensive therapies (AHT) before and after the publication of the LIFE, ALLHAT and VALUE trials between 2000 and 2005.

Methods

The Irish HSE‐PCRS prescribing database was used to identify those initiated any AHT. Any change 12 months before and after the trial publications was examined using a segmented regression analysis.

Results

There was little or no effect of any of the trials on new AHT prescribing, except for ALLHAT where there was an increase in new prescriptions for ACE inhibitors, and VALUE with a slight increase in prescriptions for calcium channel blockers.

Conclusions

Our findings show that there was little or no effect of any of the three clinical trials studied on new AHT prescribing patterns in Irish general practice. Future studies should assess any underlying barriers to implementing new evidence into clinical practice.
Interindividual variability in the concentration–effect relationship of antilymphocyte globulins—a possible influence of FcγRIIIa genetic polymorphism
Authors: Ternant David , Büchler Matthias , Bénéton Maud , Alván Gunnar , Ohresser Marc , Touchard Guy , Hurault de Ligny Bruno , Toupance Olivier , Watier Hervé , Lebranchu Yvon , Paintaud Gilles
Published: July 2007

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• There is interindividual variability in the antilymphocyte globulin (ALG) effect, but there is no pharmacokinetic–pharmacodynamic study of this subject.

• In addition, a time dependence of the pharmacokinetics of some therapeutic antibodies has been described.

• ALGs may partly act by antibody‐dependent cellular cytotoxicity (ADCC), but their mechanism of action in humans is not known.

WHAT THIS STUDY ADDS

• Horse ALG pharmacokinetics can be described using a two‐compartment model with time‐dependent central volume of distribution.

• After an initial concentration‐independent lymphocyte depletion, the concentration–effect relationship can be described using a physiological indirect response model.

• The genetic polymorphism of FcγRIIIa at position 158 may influence the ALG concentration–effect relationship and these polyclonal antibodies may therefore act by ADCC.

AIMS

Polyclonal antilymphocyte globulins (ALGs) are currently used in transplantation, but the sources of interindividual variability of their effect are poorly understood. No pharmacokinetic–pharmacodynamic (PK–PD) study of ALG is available. Moreover, the genetic polymorphism of FcγRIIIa, a receptor for the Fc portion of immunoglobulins involved in antibody‐dependent cellular cytotoxicity (ADCC), may influence their concentration–effect relationship.

METHODS

Fourteen kidney transplant patients treated by horse ALG were included in a prospective, noncomparative study. A population two‐compartment PK model including a time dependence of the central volume of distribution was developed. Total lymphocyte count was used as biomarker of effect. Concentration–effect data were described using a physiological indirect response model, combining concentration‐dependent and ‐independent inhibitions of lymphocyte input into the circulation. In addition, six kidney transplant patients in whom ALG concentrations were not available were included retrospectively. All patients were genotyped for FCGR3A.

RESULTS

Both the PK and the PK–PD model described the data satisfactorily and showed high interindividual variability. Asymptotic T1/2‐α and T1/2‐β‐values were 1.3 and 25 days, respectively. The concentration of ALG leading to a 50% inhibition of lymphocyte input (IC50) was lower in FCGR3A‐V carriers than in FCGR3A‐F/F patients (383 ± 199 vs. 593 ± 209 mg l−1, P = 0.008).

CONCLUSIONS

This is the first description of the ALG effect on lymphocyte count using PK–PD modelling. Our results show that part of the variability in their concentration–effect relationship may be explained by FcγRIIIa genetic polymorphism and therefore that horse ALG may deplete lymphocytes by ADCC.
Whose agenda? Issues of power and relationship when listening to looked‐after young people
Authors: McLeod Alison
Published: July 2007

Listening to children and young people is for social workers a legal requirement, it is also believed to lead to better outcomes and is increasingly promoted in policy. The practice of listening is, however, rarely straightforward, and this is particularly the case with socially excluded groups. As a result, the voices of the marginalized are less likely to be heard. In this paper the author draws on interview data from a research study into communication between social workers and looked‐after children for the light it sheds on why effective dialogue with disaffected young people can be hard to achieve. It is argued that much that appears unsuccessful in an interaction can be understood in terms of power plays, with young people resisting the adult's agenda and trying to impose their own. True listening to disaffected young people requires time, so that a trusting relationship can be developed. It also demands an acceptance that the adult agenda may be flawed and a willingness to consider alternative possibilities. This can challenge the preconceptions, not just of individual workers, but of agencies and of national policy‐makers.
Family support, social capital, resilience and adolescent coping
Authors: Pinkerton John , Dolan Pat
Published: July 2007

All too often young people are excluded in practice from the general policy and professional consensus that partnership and participation should underpin work with children, young people and their families. If working with troubled and troublesome young people is to be based on family support, it will require not only the clear statement of that policy but also demonstration that it can be applied in practice. Achieving that involves setting out a plausible theory of change that can be rigorously evaluated. This paper suggests a conceptual model that draws on social support theory to harness the ideas of social capital and resilience in a way that can link formal family support interventions to adolescent coping. Research with young people attending three community‐based projects for marginalized youth is used to illustrate how validated tools can be used to measure and document the detail of support, resilience, social capital and coping in young people's lives. It is also suggested that there is sufficient fit between the findings emerging from the study and the model to justify the model being more rigorously tested.
Young people's gendered interpretations of suicide and attempted suicide
Authors: Scourfield Jonathan , Jacob Nina , Smalley Nina , Prior Lindsay , Greenland Katy
Published: July 2007

This paper aims to uncover gendered interpretations of various kinds of suicidal behaviour. Its empirical basis is focus group discussions with a range of young people, including users of social work services. In support of Canetto's research, the authors found some of the young people to be associating ‘successful’ suicides with masculinity and ‘failed’ suicide attempts with femininity. These feminized suicide attempts were subject to some fairly pejorative interpretations, such as being motivated by revenge or manipulation. There was no particular pattern of viewpoints in terms of the sex of respondents. The implications of these findings for social work are discussed.
The impact of family history of diabetes on risk factors for gestational diabetes
Authors: Retnakaran Ravi , Connelly Philip W. , Sermer Mathew , Zinman Bernard , Hanley Anthony J. G.
Published: July 2007

Objective  Familial history of type 2 diabetes (FHD) represents a pathophysiologically unique risk factor for gestational diabetes (GDM), insofar as it encompasses both inherited and lifestyle elements. We thus hypothesized that the risk factors for gestational hyperglycaemia in women with FHD may differ from those in women without FHD.

Design/patients/measurements  GDM risk factors were evaluated in 90 women with FHD and in 83 women without FHD, at the time of oral glucose tolerance testing in late pregnancy.

Results  There were no significant differences between the two groups in ethnicity, prepregnancy BMI, the insulin‐sensitizing protein adiponectin, glucose tolerance status and area‐under‐the‐glucose‐curve (AUCgluc). In women with FHD, a multiple linear regression model of established GDM risk factors reconciled 35% of the variance in AUCgluc, with (i) previous GDM (t = 3·74, P = 0·0003) identified as a positive independent determinant and (ii) log adiponectin (t = –3·48, P = 0·0008) and, unexpectedly, parity (t = –3·19, P = 0·0021) emerging as negative independent covariates of AUCgluc. In contrast, in women without FHD, the same multivariate model reconciled only 15% of the variance in AUCgluc, with no significant variables identified. Interestingly, in the entire population (n = 173), parity significantly modified the relationship between FHD and AUCgluc (FHD–parity interaction: t = –2·29, P = 0·0235). Indeed, FHD was an independent determinant of AUCgluc in nulliparous women (n = 91), but not in parous women (n = 82).

Conclusion  Established risk factors for GDM are relevant in women with FHD but may not be the principal determinants of gestational hyperglycaemia in women without FHD. Moreover, FHD may be more relevant to risk of GDM in nulliparous women than in parous subjects. These findings highlight the complex relationship between FHD and gestational hyperglycaemia, and may hold implications for selective screening for GDM.
Growth hormone deficiency (GHD) in adult thalassaemic patients
Authors: Scacchi Massimo , Danesi Leila , Cattaneo Agnese , Valassi Elena , Giraldi Francesca Pecori , Argento Crocetta , D’Angelo Emanuela , Mirra Nadia , Carnelli Vittorio , Zanaboni Laura , Cappellini Maria Domenica , Cavagnini Francesco
Published: July 2007

Background and objective  Short stature and growth hormone deficiency (GHD) are frequent occurrences in thalassaemic children, while data on the prevalence of GHD in adult patients are lacking. Therefore, we elected to study the growth hormone and insulin‐like growth factor‐I (GH–IGF‐I) axis in a large group of adult thalassaemic subjects.

Design  Cross‐sectional study on the prevalence of GHD in 94 adult thalassaemic patients (69 with thalassaemia major and 25 with thalassaemia intermedia, 39 men and 55 women, aged 31·5 ± 6·8 years, on sex steroid replacement when necessary).

Methods  All patients underwent GHRH (1 µg/kg as an i.v. bolus) plus arginine (0·5 g/kg as a 30 min i.v. infusion) testing. Severe GHD was defined by GH peaks lower than 9 µg/l, whereas partial GHD was defined by GH peaks ranging from 9–16·5 µg/l. Blood samples for IGF‐I, ferritin and pseudocholinesterase measurements were collected. Urinary free cortisol (UFC) levels were also assayed.

Results  Severe GHD was demonstrated in 21 of the 94 patients (22·3%), while 18 additional patients (19·1%) displayed partial GHD. GH peaks were positively correlated with IGF‐I standard deviation score (SDS) (r = 0·22, P < 0·05), although 1 of the 21 patients with severe GHD showed normal IGF‐I SDS values, and 44 of the 55 patients with normal GH reserve displayed low IGF‐I SDS. A strong positive correlation (r = 0·48, P < 0·0001) between IGF‐I SDS and pseudocholinesterase was identified. No correlations were found between ferritin and UFC levels on the one hand and GH peaks and IGF‐I SDS on the other.

Conclusion  Findings from this study demonstrate that GHD, either partial or severe, is not a rare occurrence in adult thalassaemic patients. GHD is associated with a higher prevalence of low serum IGF‐I levels, recorded also in patients with normal GH secretion. The lack of correlation between ferritin and both GH peaks and IGF‐I SDS suggests that mechanisms additional to iron overload, whose relevance cannot however be definitely ruled out, play a role in the pathophysiology of somatotrophin‐somatomedin deficiency in this clinical condition. The positive correlation between IGF‐I SDS on the one hand and GH peaks and pseudocholinesterase values on the other hand indicates that reduced liver protidosynthetic activity, in addition to somatotrophin secretory status, is a major determinant of the impaired IGF‐I production in thalassaemia. Therefore biosynthetic GH replacement therapy in GH‐deficient thalassaemic adults is worth considering.
The mechanism of boron tolerance for maintenance of root growth in barley (Hordeum vulgare L.)
Authors: CHOI EUN‐YOUNG , KOLESIK PETER , MCNEILL ANN , COLLINS HELEN , ZHANG QISEN , HUYNH BAO‐LAM , GRAHAM ROBIN , STANGOULIS JAMES
Published: July 2007

Cultivar differences in root elongation under B toxic conditions were observed in barley (Hordeum vulgare L.). A significant increase in the length and width of the root meristematic zone (RMZ) was observed in Sahara 3771 (B tolerant) when it was grown under excessive B concentration, compared to when grown at adequate B supply. This coincided with an increase in cell width and cell numbers in the meristematic zone (MZ), whereas a significant decrease in the length and no significant effect on the width of the MZ was observed in Clipper (B intolerant) when it was grown under excessive B supply. This was accompanied by a decrease in cell numbers, but an increase in the length and width of individual cells present along the MZ. Excessive B concentrations led to a significantly lower osmotic potential within the cell sap of the root tip in SloopVic (B tolerant) and Sahara 3771, while the opposite was observed in Clipper. Enhanced sugar levels in the root tips of SloopVic were observed between 48 and 96 h after excess B was applied. This coincided with an increase in the root elongation rate and with a 2.7‐fold increase in sucrose level within mature leaf tissue. A significant decrease in reducing sugar levels was observed in the root tips of Clipper under excessive B concentrations. This coincided with significantly lower root elongation rates and lower sucrose levels in leaf tissues. Results indicate a B tolerance mechanism associated with a complex control of sucrose levels between leaf and root tip that assist in maintaining root growth under B toxicity.
A stereo imaging system for measuring structural parameters of plant canopies
Authors: BISKUP BERNHARD , SCHARR HANNO , SCHURR ULRICH , RASCHER UWE
Published: July 2007

Plants constantly adapt their leaf orientation in response to fluctuations in the environment, to maintain radiation use efficiency in the face of varying intensity and incidence direction of sunlight. Various methods exist for measuring structural canopy parameters such as leaf angle distribution. However, direct methods tend to be labour‐intensive, while indirect methods usually give statistical information on stand level rather than on individual leaves. We present an area‐based, binocular stereo system composed of commercially available components that allows three‐dimensional reconstruction of small‐ to medium‐sized canopies on the level of single leaves under field conditions. Spatial orientation of single leaves is computed with automated processes using modern, well‐established stereo matching and segmentation techniques, which were adapted for the properties of plant canopies, providing high spatial and temporal resolution (angle measurements with an accuracy of approx. ±5° and a maximum sampling rate of three frames per second). The applicability of our approach is demonstrated in three case studies: (1) the dihedral leaflet angle of an individual soybean was tracked to monitor nocturnal and daytime leaf movement showing different frequencies and amplitudes; (2) drought stress was diagnosed in soybean by quantifying changes in the zenith leaflet angle distribution; and (3) the diurnal course of the zenith leaf angle distribution of a closed soybean canopy was measured.
Mutation analysis of the genes involved in the Ras‐mitogen‐activated protein kinase (MAPK) pathway in Korean patients with Noonan syndrome
Authors: Lee S‐T , Ki C‐S , Lee HJ
Published: July 2007

Noonan syndrome (NS) is a congenital abnormality that affects multiple parts of the body. Approximately 50% of cases are caused by mutations in the PTPN11 gene. NS shares many clinical features with a group of developmental disorders including Costello syndrome and cardio‐facio‐cutaneous (CFC) syndrome. Recently, KRAS and SOS1 were identified as causative genes for NS. Moreover, mutations in several genes associated with the Ras‐mitogen‐activated protein kinase (MAPK) pathway, including HRAS, BRAF, MEK1, and MEK2 were identified in patients with Costello syndrome and CFC syndrome. Accordingly, this study carried out mutation analysis of nine genes including PTPN11, SOS1, GRB2, KRAS, HRAS, NRAS, BRAF, MEK1, and MEK2 associated with the Ras‐MAPK pathway in 14 Korean patients with NS. Seven patients were found to have mutations in the PTPN11 gene. Mutation analyses of the other genes did not reveal any disease causing mutations except for one unclassified variation in the 3′‐untranslated region of the HRAS gene (c.*1C>T). The patient’s father also had the same substitution with the normal phenotype. Therefore, this variation is believed to be either a rare polymorphism or a disease‐related variation with variable penetrance. The Ras‐MAPK pathway has now emerged as a key cascade in a group of similar developmental disorders as well as in many types of cancers. This study found that, with the exception of PTPN11, mutations in genes related to the Ras‐MAPK pathway appear to be uncommon, at least in Korean patients with NS.