bluuebunny/crossref_metadata_2025 huggingface dataset:
Compulsory hyperventilation and hypocapnia of patients with Leigh syndrome associated with SURF1 gene mutations as a cause of low serum bicarbonates
Authors: Pronicka E. , Piekutowska‐Abramczuk D. H. , Popowska E. , Pronicki M. , Karczmarewicz E. , Sykut‐Cegielskâ Y. , Taybert J.
Published: December 2002

Experimental data show that elevation of intracellular pH leads to severe lesions of brain cells. Acidification of intracellular fluid by accumulation of lactate may compensate the effect of respiratory alkalosis. Increased serum pH, and low PCO2, associated with hyperlactataemia (sometimes incorrectly called ‘acidosis’) have been reported in children with Leigh syndrome (LS).

The aim of the study was to determine whether respiratory alkalosis is characteristic of patients with LS due to SURF1 mutations. All venous blood gas data (88 samples) of 18 spontaneously breathing LS patients with recently established SURF1 mutations, hospitalized during 1986–2000, were retrospectively reviewed. The data of an affected boy who survived on a respirator for more than 3 months (79 daily samples) were analysed separately.

In spontaneously breathing patients, the data indicated that the patients had compensated or partially compensated respiratory alkalosis (pH 7.388 ± 0.060, PCO2 29.2 ± 5.7 mmHg, HCO−3 17.4 ± 3.0 mmol/L, BE −6.7 ± 3.2 mmol/L). Bicarbonate excretion was detected in urine of two examined LS cases in spite of decreased serum HCO−3. In the affected child maintained on a respirator, simple manipulation of the inspired CO2 tension to establish a normal pressure of 35–45 mmHg automatically caused an increase of serum HCO−3 concentration to a normal value of 26.3 ± 2.9 mmol/L (and BE to +2.2 ± 3.1 mmol/L), in spite of cytochrome oxidase (COX) deficiency due to a confirmed SURF1 mutation.

We suggest that respiratory alkalosis (hypocapnia) of Leigh syndrome patients with SURF1 mutations results from compulsory hyperventilation and speculate that hypocapnia may contribute to Leigh‐like brain damage in the SURF1‐deficient patients as well as in other patients presenting with Leigh‐like syndrome. The supposition that accumulation of lactate may protect the brain of LS patients from alkalosis‐related damage requires further study. Avoidance of any factors stimulating hyperventilation of LS patients and caution when attempting to correct low plasma bicarbonate are suggested.
A qualitative analysis of posttraumatic stress among Mexican victims of disaster
Authors: Norris Fran H. , Weisshaar Deborah L. , Conrad M. Lori , Diaz Eolia M. , Murphy Arthur D. , Ibañez Gladys E.
Published: December 2002

In unstructured interviews, 24 Mexicans described survivors' responses to disasters in Guadalajara, Jalisco (n = 9), Homestead, Florida (n = 6), and Puerto Angel, Oaxaca (n = 9). This analysis assessed the extent to which symptom descriptions corresponded to the 17 criterion symptoms of PTSD. Nineteen participants (79%) mentioned from 1 to 9 criterion symptoms. Event‐related distress, hypervigilance, recurrent recollections, and avoiding reminders were described most often. Only 3 criterion symptoms were never described. Twenty participants (83%) provided 109 separate expressions that could not be classified specifically as criterion symptoms. These phrases were sorted by 9 independent Mexican volunteers and cluster analyzed. Clusters composed of ataques de nervios, depression, lasting trauma, and somatic complaints provided the best description of the data.
Translating research findings to PTSD prevention: Results of a randomized–controlled pilot study
Authors: Gidron Yori , Gal Reuven , Freedman Sara , Twiser Irit , Lauden Ari , Snir Yoram , Benjamin Jonathan
Published: December 2002

Based on therapeutic studies revealing positive prognostic factors and on research findings revealing how trauma is processed, we developed the memory structuring intervention (MSI) in attempt to prevent posttraumatic stress disorder (PTSD). The MSI attempts to shift processing of traumatic memory from uncontrollable somatosensory and affective processes to more controlled linguistic and cognitive processes by providing patients organization, labeling, and causality. In a single‐blind randomized‐controlled pilot study, 17 traffic accident victims at risk for PTSD (heart rate > 94 BPM) were assigned to two MSI or two supportive‐listening control sessions. Three months later, MSI patients reported significantly less frequent intrusive, arousal, and total PTSD symptoms than controls. A replication study with a larger sample is underway.
Forced displacement in Yugoslavia: A meta‐analysis of psychological consequences and their moderators
Authors: Porter Matt , Haslam Nick
Published: December 2002

A meta‐analysis was conducted to synthesize what is known about differences in mental health between refugees and nonrefugees from the former Yugoslavia. The analysis focused on moderating effects of a variety of enduring, contextual stressors. Results indicated that refugees suffer significantly more mental health impairment than nonrefugees. The psychological consequences offorced displacement were found to vary significantly as a function of chronic stressors (e.g., locus of displacement and type of accommodation in exile) and were also associated with other factors (e.g., degree of war exposure in the nondisplaced groups, participant age, and time of data collection as reflected in year of publication). Implications for the study of refugee mental health are discussed.
Effects of writing about rape: Evaluating Pennebaker's paradigm with a severe trauma
Authors: Brown Elissa J. , Heimberg Richard G.
Published: December 2002

We examined the effect of disclosing to others an attempted or completed rape. Eighty‐five undergraduate women who acknowledged attempted or completed rape wrote about their experience and read their narratives. In a 2 × 2 design, we examined the value of writing only factual information versus factual plus emotional information, and reading to oneself versus reading aloud to another woman. Before and 1‐month after the task, symptoms of dysphoria, social anxiety, and posttraumatic stress disorder were assessed. Greater detail and a moderate level of personalization in the description of the trauma were associated with decreased symptoms of dysphoria and social anxiety. Neither the nature of the writing task nor the presence of another woman predicted degree of symptom reduction.
The Neurobiology of Dopamine Signaling
Authors: Greengard Paul
Published: December 2002

The biochemistry of synaptic transmission, especially the neurobiology of dopamine signaling, is discussed.
The Relationship Between the Reference Value of Erythrocyte Sedimentation Rate and Geographical Factors
Authors: Miao Ge
Published: December 2002

In order to provide a scientific basis for a unified standard of the reference value of erythrocyte sedimentation rate (ESR) in China, the reference value of healthy people's ESR has been collected. The relationship between the reference value of ESR and geographical factors is examined in this paper. Altitude is the most important factor affecting the reference value of ESR, which decreases with increasing altitude; the relationship is quite significant. The method of stepwise regression analysis was used to deduce two regression equations: Ŷ1=12.08–.00222X1+0.00114X5 ± 2.95, Ŷ2=18.81–0.00323X1+0.239X4 ± 4.70. If the geographical factor value of a particular area in China is known, the reference value of ESR of Chinese people can be calculated by means of the regression equations. Furthermore, the dependence on geographical factors in China can be classified as six districts: Qingzang, Southwest, Northwest, Southeast, North and Northeast.
Stress-Induced Changes in Ubiquinone Concentration and Alternative Oxidase in Plant Mitochondria
Authors: Popov Vasily N. , Purvis Albert C. , Skulachev Vladimir P. , Wagner Anneke M.
Published: December 2002

We have investigated the influence of stress conditions such as incubation at 4°C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4°C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.
Fusion and Infection of Influenza and Sendai Viruses as Modulated by Dextran Sulfate: A Comparative Study
Authors: Ramalho-Santos João , Pedroso de Lima Maria C.
Published: December 2002

We have directly compared the effect of two types of dextran sulfate with distinct molecular weights (500 kDa and 5 kDa) on the fusion activity and infectivity of both Sendai and influenza viruses, two lipid-enveloped viruses that differ in their routes of entry into target cells. To correlate membrane merging and infectivity MDCK cells were used as targets for the viruses in both approaches. In either case pronounced inhibition of virus–cell interactions by dextran sulfate was only observed at low pH, even though Sendai virus fuses maximally at pH 7.4. Although membrane merging could not be fully abolished, the inhibitory effect was always greater when the higher molecular weight dextran sulfate was used. The presence of this residual fusion activity, that could not be reduced even with high concentrations of agent, suggests that a limited number of binding sites for dextran sulfate may exist on the viral envelopes. The compounds also inhibited fusion of bound virions, and all results could be reproduced using erythrocyte ghosts as target membranes in the fusion assay, instead of MDCK cells. In agreement with these observations only the infectivity of influenza virus (which requires a low pH-dependent step to enter target cells) was affected by dextran sulfate, again the higher molecular weight compound showing a more pronounced inhibitory effect.
Barbara Cannon's Data on the UCP1-Ablated Mice: “Non-Cannonical” Point of View
Authors: Skulachev Vladimir P.
Published: December 2002

The data of Cannon and co-workers on UCP1-ablated mice are interpreted assuming that UCP2 and UCP3 are involved in thermoregulation as fatty acid-dependent uncouplers although they are not sufficient, in the absence of UCP1, for long term maintenance of normal body temperature of mice after sudden and strong decrease in the ambient temperature. I would like to suggest that in brown fat of control mice, UCP1 is present in an amount higher than UCP2 and 3 and, therefore, is able to cause (a) some fatty acid-mediated decrease in proton motive force in resting state and, hence, (b) oxidation of CoQH2 to CoQ which is shown by Klingenberg and coworkers to be cofactor for UCPs. This results in strong uncoupling and thermogenesis mediated by UCP1, 2 and 3. In the UCP1-ablated mice, activity of UCP2 and 3 appears to be insufficient to induce CoQH2 oxidation in resting brown fat mitochondria, which results in hypothermia.
Effects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA
Authors: O'Neill Melanie A. , Barton Jacqueline K.
Published: December 2002

Mechanistic models of charge transfer (CT) in macromolecules often focus on CT energetics and distance as the chief parameters governing CT rates and efficiencies. However, in DNA, features unique to the DNA molecule, in particular, the structure and dynamics of the DNA base stack, also have a dramatic impact on CT. Here we probe the influence of subtle structural variations on base–base CT within a DNA duplex by examining photoinduced quenching of 2-aminopurine (Ap) as a result of hole transfer (HT) to guanine (G). Photoexcited Ap is used as a dual reporter of variations in base stacking and CT efficiency. Significantly, the unique features of DNA, including the strandedness and directional asymmetry of the double helix, play a defining role in CT efficiency. For an (AT)
n
bridge, the orientation of the base pairs is critical; the yield of intrastrand HT is markedly higher through (A)
n
compared with (T)
n
bridges, whereas HT via intrastrand pathways is more efficient than through interstrand pathways. Remarkably, for reactions through the same DNA bridge, over the same distance, and with the same driving force, HT from photoexcited Ap to G in the 5′ to 3′ direction is more efficient and less dependent on distance than HT from 3′ to 5′. We attribute these differences in HT efficiency to variations in base–base coupling within the DNA assemblies. Thus base–base coupling is a critical parameter in DNA CT and strongly depends on subtle structural nuances of duplex DNA.
Premonitory patterns of seismicity months before a large earthquake: Five case histories in Southern California
Authors: Keilis-Borok V. I. , Shebalin P. N. , Zaliapin I. V.
Published: December 2002

This article explores the problem of short-term earthquake prediction based on spatio-temporal variations of seismicity. Previous approaches to this problem have used precursory seismicity patterns that precede large earthquakes with “intermediate” lead times of years. Examples include increases of earthquake correlation range and increases of seismic activity. Here, we look for a renormalization of these patterns that would reduce the predictive lead time from years to months. We demonstrate a combination of renormalized patterns that preceded within 1–7 months five large (
M
≥ 6.4) strike-slip earthquakes in southeastern California since 1960. An algorithm for short-term prediction is formulated. The algorithm is self-adapting to the level of seismicity: it can be transferred without readaptation from earthquake to earthquake and from area to area. Exhaustive retrospective tests show that the algorithm is stable to variations of its adjustable elements. This finding encourages further tests in other regions. The final test, as always, should be advance prediction. The suggested algorithm has a simple qualitative interpretation in terms of deformations around a soon-to-break fault: the blocks surrounding that fault began to move as a whole. A more general interpretation comes from the phenomenon of self-similarity since our premonitory patterns retain their predictive power after renormalization to smaller spatial and temporal scales. The suggested algorithm is designed to provide a short-term approximation to an intermediate-term prediction. It remains unclear whether it could be used independently. It seems worthwhile to explore similar renormalizations for other premonitory seismicity patterns.
Autoantibodies against α-MSH, ACTH, and LHRH in anorexia and bulimia nervosa patients
Authors: Fetissov Sergueï O. , Hallman Jarmila , Oreland Lars , af Klinteberg Britt , Grenbäck Eva , Hulting Anna-Lena , Hökfelt Tomas
Published: December 2002

The hypothalamic arcuate nucleus is involved in the control of energy intake and expenditure and may participate in the pathogenesis of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). Two systems are of particular interest in this respect, synthesizing α-melanocyte-stimulating hormone (α-MSH) and synthesizing neuropeptide Y, respectively. We report here that 42 of 57 (74%) AN and/or BN patients studied had in their plasma Abs that bind to melanotropes and/or corticotropes in the rat pituitary. Among these sera, 8 were found to bind selectively to α-MSH-positive neurons and their hypothalamic and extrahypothalamic projections as revealed with immunostaining on rat brain sections. Adsorption of these sera with α-MSH peptide abolished this immunostaining. In the pituitary, the immunostaining was blocked by adsorption with α-MSH or adrenocorticotropic hormone. Additionally, 3 AN/BN sera bound to luteinizing hormone-releasing hormone (LHRH)-positive terminals in the rat median eminence, but only 2 of them were adsorbed with LHRH. In the control subjects, 2 of 13 sera (16%) displayed similar to AN/BN staining. These data provide evidence that a significant subpopulation of AN/BN patients have autoantibodies that bind to α-MSH or adrenocorticotropic hormone, a finding pointing also to involvement of the stress axis. It remains to be established whether these Abs interfere with normal signal transduction in the brain melanocortin circuitry/LHRH system and/or in other central and peripheral sites relevant to food intake regulation, to what extent such effects are related to and/or could be involved in the pathophysiology or clinical presentation of AN/BN, and to what extent increased stress is an important factor for production of these autoantibodies.
Characterization of the intergenic RNA profile at
abdominal-A
and
Abdominal-B
in the
Drosophila
bithorax complex
Authors: Bae Esther , Calhoun Vincent C. , Levine Michael , Lewis Edward B. , Drewell Robert A.
Published: December 2002

The correct spatial expression of two
Drosophila
bithorax complex (BX-C) genes,
abdominal-A
(
abdA
) and
Abdominal-B
(
AbdB
), is dependent on the 100-kb intergenic
infraabdominal
(
iab
) region. The
iab
region is known to contain a number of different domains (
iab2
through
iab8
) that harbor cis-regulatory elements responsible for directing expression of
abdA
and
AbdB
in the second through eighth abdominal segments. Here, we use
in situ
hybridization to perform high-resolution mapping of the transcriptional activity in the
iab
control regions. We show that transcription of the control regions themselves is abundant and precedes activation of the
abdA
and
AbdB
genes. As with the homeotic genes of the BX-C, the transcription patterns of the RNAs from the
iab
control regions demonstrate colinearity with the sequence of the
iab
regions along the chromosome and the domains in the embryo under the control of the specific
iab
regions. These observations suggest that the intergenic RNAs may play a role in initiating cis regulation at the BX-C early in development.
Cleavage of polycystin-1 requires the receptor for egg jelly domain and is disrupted by human autosomal-dominant polycystic kidney disease 1-associated mutations
Authors: Qian Feng , Boletta Alessandra , Bhunia Anil K. , Xu Hangxue , Liu Lijuan , Ahrabi Ali K. , Watnick Terry J. , Zhou Fang , Germino Gregory G.
Published: December 2002

Polycystin-1 plays an essential role in renal tubular morphogenesis, and disruption of its function causes cystogenesis in human autosomal-dominant polycystic kidney disease (ADPKD). We demonstrated that polycystin-1 undergoes cleavage at G protein coupled receptor proteolytic site in a process that requires the receptor for egg jelly domain. Most of the N-terminal fragment remains tethered at the cell surface, although a small amount is secreted.PKD1-associated mutations in the receptor for egg jelly domain disrupt cleavage, abolish the ability of polycystin-1 to activate signal transducer and activator of transcription-1, and induce tubulogenesisin vitro. We conclude that the cleavage of polycystin-1 is likely essential for its biologic activity.
Directed evolution of a genetic circuit
Authors: Yokobayashi Yohei , Weiss Ron , Arnold Frances H.
Published: December 2002

The construction of artificial networks of transcriptional control elements in living cells represents a new frontier for biological engineering. However, biological circuit engineers will have to confront their inability to predict the precise behavior of even the most simple synthetic networks, a serious shortcoming and challenge for the design and construction of more sophisticated genetic circuitry in the future. We propose a combined rational and evolutionary design strategy for constructing genetic regulatory circuits, an approach that allows the engineer to fine-tune the biochemical parameters of the networks experimentally
in vivo
. By applying directed evolution to genes comprising a simple genetic circuit, we demonstrate that a nonfunctional circuit containing improperly matched components can evolve rapidly into a functional one. In the process, we generated a library of genetic devices with a range of behaviors that can be used to construct more complex circuits.
Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron
Authors: D'Autréaux Benoît , Touati Danièle , Bersch Beate , Latour Jean-Marc , Michaud-Soret Isabelle
Published: December 2002

Ferric uptake regulation protein (Fur) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. The function of Fur is not limited to iron homeostasis. A wide variety of genes involved in various mechanisms such as oxidative and acid stresses are under Fur control. Flavohemoglobin (Hmp) is an NO-detoxifying enzyme induced by NO and nitrosothiol compounds. Fur recently was found to regulate
hmp
in
Salmonella typhimurium
, and in
Escherichia coli
, the iron-chelating agent 2,2′-dipyridyl induces
hmp
expression. We now establish direct inhibition of
E. coli
Fur activity by NO. By using chromosomal Fur-regulated
lacZ
reporter fusion in
E. coli
, Fur activity is switched off by NO at micromolar concentration.
In vitro
Fur DNA-binding activity, as measured by protection of restriction site in aerobactin promoter, is directly sensitive to NO. NO reacts with Fe
II
in purified FeFur protein to form a S = 1/2 low-spin FeFur–NO complex with a
g
= 2.03 EPR signal. Appearance of the same EPR signal in NO-treated cells links nitrosylation of the iron with Fur inhibition. The nitrosylated Fur protein is still a dimer and is stable in anaerobiosis but slowly decays in air. This inhibition probably arises from a conformational switch, leading to an inactive dimeric protein. These data establish a link between control of iron metabolism and the response to NO effects.
Satellite imagery characterizes local animal reservoir populations of Sin Nombre virus in the southwestern United States
Authors: Glass Gregory E. , Yates Terry L. , Fine Joshua B. , Shields Timothy M. , Kendall John B. , Hope Andrew G. , Parmenter Cheryl A. , Peters C. J. , Ksiazek Thomas G. , Li Chung-Sheng , Patz Jonathan A. , Mills James N.
Published: December 2002

The relationship between the risk of hantaviral pulmonary syndrome (HPS), as estimated from satellite imagery, and local rodent populations was examined. HPS risk, predicted before rodent sampling, was highly associated with the abundance of
Peromyscus maniculatus
, the reservoir of Sin Nombre virus (SNV).
P. maniculatus
were common in high-risk sites, and populations in high-risk areas were skewed toward adult males, the subclass most frequently infected with SNV. In the year after an El Niño Southern Oscillation (ENSO), captures of
P. maniculatus
increased only in high-risk areas. During 1998, few sites had infected mice, but by 1999, 18/20 of the high-risk sites contained infected mice and the crude prevalence was 30.8%. Only 1/18 of the low-risk sites contained infected rodents, and the prevalence of infection was lower (8.3%). Satellite imagery identified environmental features associated with SNV transmission within its reservoir population, but at least 2 years of high-risk conditions were needed for SNV to reach high prevalence. Areas with persistently high-risk environmental conditions may serve as refugia for the survival of SNV in local mouse populations.
A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling
Authors: Karlström Helena , Beatus Paul , Dannaeus Karin , Chapman Gavin , Lendahl Urban , Lundkvist Johan
Published: December 2002

Notch receptors are single transmembrane receptors that contain a large number of epidermal growth factor-like repeats (EGF repeats) in their extracellular domains. Mutations in the EGF repeats of the human Notch 3 receptor lead to the vascular dementia disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The vast majority of CADASIL mutations are missense mutations removing or inserting cysteine residues in the EGF repeats, but it is not yet clear whether these mutations primarily affect receptor trafficking, maturation, and/or signaling. To address this issue, we have generated and analyzed stable cell lines expressing either wild-type murine Notch 3 (mNotch 3) or the mutant mNotch 3
R142C
, which corresponds to the prevalent CADASIL form of Notch 3, Notch 3
R141C
in humans. We find that a lower proportion of mNotch 3
R142C
is expressed in the site 1-cleaved configuration, and that reduced amounts of mNotch 3
R142C
appear at the cell surface, as compared with wild-type mNotch 3. This observation is accompanied by a higher propensity for mNotch 3
R142C
to form intracellular aggregates, which may be a result of increased accumulation or slowed transport in the secretory pathway. In contrast to the impaired cell surface expression, mNotch 3
R142C
signals equally well in response to Delta 1 and Jagged 1 as wild-type mNotch 3. Taken together, these data suggest that trafficking and localization rather than signaling of mNotch 3 are affected in mNotch 3
R142C
.
Noninvasive real-time imaging of apoptosis
Authors: Laxman Bharathi , Hall Daniel E. , Bhojani Mahaveer Swaroop , Hamstra Daniel A. , Chenevert Thomas L. , Ross Brian D. , Rehemtulla Alnawaz
Published: December 2002

Strict coordination of proliferation and programmed cell death (apoptosis) is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemia/reperfusion injury, cancer, autoimmune disease, among others. Objective and quantitative noninvasive imaging of apoptosis would be a significant advance for rapid and dynamic screening as well as validation of experimental therapeutic agents. Here, we report the development of a recombinant luciferase reporter molecule that when expressed in mammalian cells has attenuated levels of reporter activity. In cells undergoing apoptosis, a caspase-3-specific cleavage of the recombinant product occurs, resulting in the restoration of luciferase activity that can be detected in living animals with bioluminescence imaging. The ability to image apoptosis noninvasively and dynamically over time provides an opportunity for high-throughput screening of proapoptotic and antiapoptotic compounds and for target validation
in vivo
in both cell lines and transgenic animals.
Genomic diversity of enterohemorrhagic
Escherichia coli
O157 revealed by whole genome PCR scanning
Authors: Ohnishi Makoto , Terajima Jun , Kurokawa Ken , Nakayama Keisuke , Murata Takahiro , Tamura Kazumichi , Ogura Yoshitoshi , Watanabe Haruo , Hayashi Tetsuya
Published: December 2002

Enterohemorrhagic
Escherichia coli
O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse
Xba
I-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.
Protection against ischemic brain injury by protein therapeutics
Authors: Asoh Sadamitsu , Ohsawa Ikuroh , Mori Takashi , Katsura Ken-ichiro , Hiraide Tomoharu , Katayama Yasuo , Kimura Megumi , Ozaki Daiya , Yamagata Kumi , Ohta Shigeo
Published: December 2002

Preventing massive cell death is an important therapeutic strategy for various injuries and disorders. Protein therapeutics have the advantage of delivering proteins in a short period. We have engineered the antiapoptotic
bcl-x
gene to generate the super antiapoptotic factor, FNK, with a more powerful cytoprotective activity. In this study, we fused the protein transduction domain (PTD) of the HIV/Tat protein to FNK and used the construct in an animal model of ischemic brain injury. When added into culture media of human neuroblastoma cells and rat neocortical neurons, PTD-FNK rapidly transduced into cells and localized to mitochondria within 1 h. It protected the neuroblastomas and neurons against staurosporine-induced apoptosis and glutamate-induced excitotoxicity, respectively. The cytoprotective activity of PTD-FNK was found at concentrations as low as 0.3 pM. Additionally, PTD-FNK affected the cytosolic movement of calcium ions, which may relate to its neuroprotective action. Immunohistochemical analysis revealed that myc-tagged PTD-FNK (PTD-myc-FNK) injected i.p. into mice can have access into brain neurons. When injected i.p. into gerbils, PTD-FNK prevented delayed neuronal death in the hippocampus caused by transient global ischemia. These results suggest that PTD-FNK has a potential for clinical utility as a protein therapeutic strategy to prevent cell death in the brain.
Anaerobic enzyme⋅substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase
Authors: Steiner Roberto A. , Kalk Kor H. , Dijkstra Bauke W.
Published: December 2002

Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two C—C bonds are broken and concomitantly carbon monoxide is released. The x-ray structures of
Aspergillus
japonicus
2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-Å resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro
164
, a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp
3
character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu
73
coordinates the copper through its Oɛ1 atom. The short distance of about 2.55 Å between its Oɛ2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu
73
can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O
2
binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.
Differential lipid biosynthesis underlies a tradeoff between reproduction and flight capability in a wing-polymorphic cricket
Authors: Zhao Zhangwu , Zera Anthony J.
Published: December 2002

The biochemical basis of life-history tradeoffs is a poorly studied aspect of life-history evolution. We used radiotracer and endocrine approaches to investigate the extent to which morphs of a wing-polymorphic insect differ in the biosynthesis of lipid classes important for dispersal capability vs. reproduction (ovarian growth). The flight-capable genotype ofGryllus firmusbiosynthesized a greater amount of total lipid and triglyceride (main flight fuel), which was preferentially allocated to somatic tissue during early adulthood. By contrast, the flightless genotype biosynthesized a significantly greater amount of phospholipid (important in egg development), which was preferentially allocated to ovaries. Topical application of a juvenile-hormone mimic to the flight-capable morph caused it to express all aspects of lipid metabolism seen in the flightless morph. Differences in biosynthesis between morphs (i) occur coincident with 100–400% greater ovarian growth in the flightless morph, (ii) result from alterations of bothde novobiosynthesis of fatty acid and downstream partitioning of fatty acids into triglyceride vs. phospholipid, and (iii) possibly result from genetically polymorphic hormonal regulators with negative pleiotropic effects on lipid biosynthesis and ovarian growth. The present study provides direct documentation of genetically based alterations ofin vivoflux through pathways of intermediary metabolism leading to the differential production of end products central to the specialization of phenotypes for alternate life histories.
Carotenoid to chlorophyll energy transfer in the peridinin–chlorophyll-
a
–protein complex involves an intramolecular charge transfer state
Authors: Zigmantas Donatas , Hiller Roger G. , Sundström Villy , Polívka Tomáš
Published: December 2002

Carotenoids are, along with chlorophylls, crucial pigments involved in light-harvesting processes in photosynthetic organisms. Details of carotenoid to chlorophyll energy transfer mechanisms and their dependence on structural variability of carotenoids are as yet poorly understood. Here, we employ femtosecond transient absorption spectroscopy to reveal energy transfer pathways in the peridinin–chlorophyll-
a
–protein (PCP) complex containing the highly substituted carotenoid peridinin, which includes an intramolecular charge transfer (ICT) state in its excited state manifold. Extending the transient absorption spectra toward near-infrared region (600–1800 nm) allowed us to separate contributions from different low-lying excited states of peridinin. The results demonstrate a special light-harvesting strategy in the PCP complex that uses the ICT state of peridinin to enhance energy transfer efficiency.
Site-directed recombination via bifunctional PNA–DNA conjugates
Authors: Rogers Faye A. , Vasquez Karen M. , Egholm Michael , Glazer Peter M.
Published: December 2002

Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson–Crick base pairing and can form triple helices via Hoogsteen binding to DNA/PNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA–DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background. Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA–DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNA-induced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA–DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.
Reversible infertility in male mice after oral administration of alkylated imino sugars: A nonhormonal approach to male contraception
Authors: van der Spoel Aarnoud C. , Jeyakumar Mylvaganam , Butters Terry D. , Charlton Harry M. , Moore Harry D. , Dwek Raymond A. , Platt Frances M.
Published: December 2002

During mammalian spermatogenesis, male germ cells undergo a dramatic transformation, which includes a change of shape, nuclear condensation, and development of specialised structures, such as an acrosome, and a flagellum with a mitochondrial sheath. We have found a previously undescribed pharmacological approach to intervene in these events. After oral administration of the alkylated imino sugarN-butyldeoxynojirimycin (NB-DNJ) to mice, epididymal spermatozoa displayed a spectrum of abnormal head shapes, and acrosomal antigens were mostly absent or displayed irregular patterns. In addition, the mitochondria of these cells often had an aberrant morphology, and were arranged in relatively short and wide mitochondrial sheaths. The motility of the affected spermatozoa was severely impaired. After 3 weeks of administration ofNB-DNJ, male mice became sterile, and regained their fertility during the fourth week off drug. TheNB-DNJ-induced infertility was not associated with a reduction in the serum testosterone level. Biochemically, the capacity of imino sugars to impair spermatogenesis was associated with their potential to attenuate the biosynthesis of glucosylceramide-based sphingolipids. Our study reveals that male fertility is affected by partial glycosphingolipid depletion, or, alternatively, by a distinct as yet unidentified property that is shared by alkylated imino sugars that inhibit glucosylceramide biosynthesis. These compounds therefore may be new leads in the development of a male contraceptive, especially becauseNB-DNJ has already been through extensive evaluation in various mammals, including man.
Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts
Authors: Rydberg Bjorn , Game John
Published: December 2002

Misincorporated ribonucleotides in DNA will cause DNA backbone distortion and may be targeted by DNA repair enzymes. Using double-stranded oligonucleotide probes containing a single ribose, we demonstrate a robust activity in human, yeast, and
Escherichia coli
cell-free extracts that nicks 5′ of the ribose. The human and yeast extracts also make a subsequent cut 3′ of the ribonucleotide releasing a ribonucleotide monophosphate. The resulting 1-nt gap is an ideal substrate for polymerase and ligase to complete a proposed repair sequence that effectively replaces the ribose with deoxyribose. Screening of yeast deletion mutant cells reveals that the initial nick is made by RNase H(35), a RNase H type 2 enzyme, and the second cut is made by Rad27p, the yeast homologue of human FEN-1 protein. RNase H type 2 enzymes are present in all kingdoms of life and are evolutionarily well conserved. We knocked out the corresponding
rnhb
gene in
E. coli
and show that extracts from this strain lack the nicking activity. Conversely, a highly purified archaeal RNase HII type 2 protein has a pronounced activity. To study substrate specificity, extracts were made from a yeast double mutant lacking the other main RNase H enzymes [RNase H1 and RNase H(70)], while maintaining RNase H(35). It was found that a single ribose is preferred as substrate over a stretch of riboses, further strengthening a proposed role of this enzyme in the repair of misincorporated ribonucleotides rather than (or in addition to) processing RNA/DNA hybrid molecules.
Trbp111 selectively binds a noncovalently assembled tRNA-like structure
Authors: Kushiro Tetsuo , Schimmel Paul
Published: December 2002

Transfer RNAs are key components of the genetic code by virtue of aminoacylation reactions whereby each amino acid is linked to the tRNA that bears the anticodon for the attached amino acid. The L-shaped tRNA structure contains two domains connected at right angles through a corner formed from tertiary interactions involving loops of each domain. Some evidence suggests that the domains arose separately and eventually were fused into a single covalent structure. In this scenario, the present-day tRNA possibly developed through a noncovalently assembled heterodimeric intermediate. Trbp111 is an ancient structure-specific tRNA binding protein that interacts specifically with the outside corner of the L-shaped molecule. Plausibly, this protein could act as a chaperone to cover and protect the fragile corner and thereby have a historical role in the development of tRNA. Here we show that Trbp111 interacts with a noncovalently assembled tRNA-like structure, under conditions where it does not interact with individual tRNA domains. Trbp111 binding specifically requires formation of the tRNA-like corner. In a mixture of RNA domains, it selects those that can make the L-like structure. Thus, cofactors such as Trbp111 have the capacity to help assemble and stabilize RNA dimers that are tRNA-like.
A role for PKC-ε in FcγR-mediated phagocytosis by RAW 264.7 cells
Authors: Larsen Elaine C. , Ueyama Takehiko , Brannock Pamela M. , Shirai Yasuhito , Saito Naoaki , Larsson Christer , Loegering Daniel , Weber Peter B. , Lennartz Michelle R.
Published: December 2002

Protein kinase C (PKC) plays a prominent role in immune signaling, and the paradigms for isoform selective signaling are beginning to be elucidated. Real-time microscopy was combined with molecular and biochemical approaches to demonstrate a role for PKC-ε in Fcγ receptor (FcγR)–dependent phagocytosis. RAW 264.7 macrophages were transfected with GFP-conjugated PKC isoforms, and GFP movement was followed during phagocytosis of fluorescent IgG–opsonized beads. PKC-ε, but not PKC-δ, concentrated around the beads. PKC-ε accumulation was transient; apparent as a “flash” on target ingestion. Similarly, endogenous PKC-ε was specifically recruited to the nascent phagosomes in a time-dependent manner. Overexpression of PKC-ε, but not PKC-α, PKC-δ, or PKC-γ enhanced bead uptake 1.8-fold. Additionally, the rate of phagocytosis in GFP PKC-ε expressors was twice that of cells expressing GFP PKC-δ. Expression of the regulatory domain (εRD) and the first variable region (εV1) of PKC-ε inhibited uptake, whereas the corresponding PKC-δ region had no effect. Actin polymerization was enhanced on expression of GFP PKC-ε and εRD, but decreased in cells expressing εV1, suggesting that the εRD and εV1 inhibition of phagocytosis is not due to effects on actin polymerization. These results demonstrate a role for PKC-ε in FcγR-mediated phagocytosis that is independent of its effects on actin assembly.
Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1
Authors: Denker Sheryl P. , Barber Diane L.
Published: December 2002

Directed cell movement is a multi-step process requiring an initial spatial polarization that is established by asymmetric stimulation of Rho GTPases, phosphoinositides (PIs), and actin polymerization. We report that the Na-H exchanger isoform 1 (NHE1), a ubiquitously expressed plasma membrane ion exchanger, is necessary for establishing polarity in migrating fibroblasts. In fibroblasts, NHE1 is predominantly localized in lamellipodia, where it functions as a plasma membrane anchor for actin filaments by its direct binding of ezrin/radixin/moesin (ERM) proteins. Migration in a wounding assay was impaired in fibroblasts expressing NHE1 with mutations that independently disrupt ERM binding and cytoskeletal anchoring or ion transport. Disrupting either function of NHE1 impaired polarity, as indicated by loss of directionality, mislocalization of the Golgi apparatus away from the orientation of the wound edge, and inhibition of PI signaling. Both functions of NHE1 were also required for remodeling of focal adhesions. Most notably, lack of ion transport inhibited de-adhesion, resulting in trailing edges that failed to retract. These findings indicate that by regulating asymmetric signals that establish polarity and by coordinating focal adhesion remodeling at the cell front and rear, cytoskeletal anchoring by NHE1 and its localized activity in lamellipodia act cooperatively to integrate cues for directed migration.
Reconstruction of DNA sequencing by hybridization
Authors: Zhang Ji-Hong , Wu Ling-Yun , Zhang Xiang-Sun
Published: December 2002

Motivation: It is widely recognized that the hybridization process is prone to errors and that the future of DNA sequencing by hybridization is predicated on the ability to successfully cope with such errors. However, the occurrence of hybridization errors results in the computational difficulty of the reconstruction of DNA sequencing by hybridization. The reconstruction problem of DNA sequencing by hybridization with errors is a strongly NP-hard problem. So far the problem has not been solved well.

Results: In this paper, a new approach is presented to solve the reconstruction problem of DNA sequencing by hybridization, which realizes the computational part of the SBH experiment. The proposed algorithm accepts both the negative and positive errors. The computational experiments show that the algorithm behaves satisfactorily, especially for the case with k-tuple repetitions and positive errors.

Contact: zhjihong@amss.ac.cn

* To whom correspondence should be addressed.
Simulating complex traits influenced by genes with
fuzzy-valued effects in pedigreed populations
Authors: Carleos C. , Rodriguez F. , Lamelas H. , Baro J.A.
Published: December 2002

Motivation: Methods involving fuzzy theory have been rarely applied to genetics. We present an open platform for experimentation with fuzzy numbers as a tool to represent imprecise phenotypes in genetic modeling.

Results: A C++ library for simulation of genetic information transmission is introduced. The study of genetic linkage was its first goal, though a design so general as possible has been meant. Fuzzy-valued phenotypes are handled by means of fuzzy numbers.

Availability: ftp://carleos.etsiig.uniovi.es/pub/falinftp://fisher.ciencias.uniovi.es/pub/falinftp://bellman.ciencias.uniovi.es/pub/falin Licensed under the GNU General Public License version 2 (see http://www.gnu.org/licenses/gpl.html)

Contact: carleos@pinon.ccu.uniovi.es

* To whom correspondence should be addressed.
An integrated genetic data environment (GDE)-based LINUX interface
for analysis of HIV-1 and other microbial sequences
Authors: De Oliveira T. , Miller R. , Tarin M. , Cassol S.
Published: December 2002

Motivation: Sequence databases encode a wealth of information needed to develop improved vaccination and treatment strategies for the control of HIV and other important pathogens. To facilitate effective utilization of these datasets, we developed a user-friendly GDE-based LINUX interface that reduces input/output file formatting.

Design and Results: GDE was adapted to the Linux operating system, bioinformatics tools were integrated with microbe-specific databases, and up-to-date GDE menus were developed for several clinically important viral, bacterial and parasitic genomes. Each microbial interface was designed for local access and contains Genbank, BLAST-formatted and phylogenetic databases.

Availability: GDE-Linux is available for research purposes by direct application to the corresponding author. Application-specific menus and support files can be downloaded from (http://www.bioafrica.net)

Contact: toliveira@mrc.ac.za

* To whom correspondence should be addressed.