bluuebunny/crossref_metadata_2025 huggingface dataset:
Two new balancer chromosomes on mouse chromosome 4 to facilitate functional annotation of human chromosome 1p
Authors: Nishijima Ichiko , Mills Alea , Qi Yi , Mills Michael , Bradley Allan
Published: July 2003

Summary: To facilitate genetic screens to identify and maintain recessive mutations that map to the short arm of human chromosome 1, we have utilized chromosome engineering to generate two mouse strains that carry large inversions on the distal region of mouse chromosome 4. The inversion intervals are 16 and 22 cM in size together they cover approximately half of chromosome 4. Since recombination between the wild‐type and inversion chromosomes does not occur within these inversion intervals, mutant alleles of genes mapping to this region can be identified and maintained. Therefore, these inversion chromosomes work as balancer chromosomes. These inversions have the additional advantage that they are tagged with genes encoding the visible coat color markers tyrosinase and agouti, and therefore the dosage of the inversion chromosome (+/+, Inv/+, Inv/Inv) can be visually recognized. These inversion strains will be extremely useful for mutagenesis screens that focus on functional annotation of human chromosome 1p. genesis 36:142–148, 2003. © 2003 Wiley‐Liss, Inc.
Patched 1 conditional null allele in mice
Authors: Ellis Tammy , Smyth Ian , Riley Emily , Graham Scott , Elliot Kate , Narang Monica , Kay Graham F. , Wicking Carol , Wainwright Brandon
Published: July 2003

Summary: The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function. genesis 36:158–161, 2003. © 2003 Wiley‐Liss, Inc.
Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice
Authors: Gilchrist Derek S. , Ure Jan , Hook Lilian , Medvinsky Alexander
Published: July 2003

Summary: Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These “activated clones” were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental “silent” clones were then used for generating mice. Upon Cre‐mediated activation in ovo tissues tested from these mice express EGFP. Long‐term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony‐forming units (CFU‐S) reside within the EGFP positive fraction. Most in vitro colony‐forming units (CFU‐Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre‐mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue‐specific Cre‐expressing mice. genesis 36:168–176, 2003. © 2003 Wiley‐Liss, Inc.
A retained selection cassette increases reporter gene expression without affecting tissue distribution in SPI3 knockout/GFP knock‐in mice
Authors: Scarff Katrina L. , Ung Kheng S. , Sun Jiuru , Bird Phillip I.
Published: July 2003

Summary: The human serpin, proteinase inhibitor 6 (PI‐6/SERPINB6), is a protease inhibitor expressed in many tissues. It inhibits a large number of proteases, including cathepsin G in granulocytes and monocytes. To determine the temporal and spatial distribution of PI‐6, mice were generated in which exon 2 of the PI‐6 ortholog SPI3 (Serpinb6) was replaced with a green fluorescent protein (GFP) reporter gene. This placed GFP under the control of the regulatory elements and initiation codon of the SPI3 gene. The neomycin selection cassette was flanked by loxP sites to allow excision from the targeted allele. GFP expression in heterozygous and SPI3‐deficient mice accurately reflected the tissue distribution of SPI3 in all organs tested and allowed precise comparisons of expression levels. Interestingly, retention of the neomycin cassette in targeted mice resulted in 2–10‐fold increases of GFP in leukocytes, but without affecting tissue‐specific expression patterns. This is the first example of selection cassette retention specifically increasing reporter gene expression in targeted mice and reinforces the view that selection cassettes must be removed to avoid confounding effects on reporter gene expression patterns. genesis 36:149–157, 2003. © 2003 Wiley‐Liss, Inc.
Mutational analysis of loxP sites for efficient Cre‐mediated insertion into genomic DNA
Authors: Thomson James G. , Rucker Edmund B. , Piedrahita Jorge A.
Published: July 2003

Summary: The Cre/loxP system has been used in transgenic models primarily to excise DNA flanked by loxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant loxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing λ integrase, mutant loxP sites were placed at the E. coli attB site and the excision‐insertion ratios of incoming DNA plasmids carrying a second, complementary mutant loxP site were determined. Comparison of 50 mutant loxP sites combinations to the native loxP site revealed that mutations to the inner 6 bp of the Cre binding domain severely inhibited recombination, while those in the outer 8 bps were more tolerated. The most efficient loxP combinations resulted in 1,421‐fold and 1,529‐fold increases in relative integration rates over wild‐type loxP sites. These loxP mutants could be exploited for site‐directed “tag and insert” recombination experiments. genesis 36:162–167, 2003. © 2003 Wiley‐Liss, Inc.
Modulation of neural activities by enhanced local selection in the processing of compound stimuli
Authors: Han Shihui , He Xun
Published: July 2003

The global precedence effect refers to the findings that responses are faster to a global structure than to its local parts and local responses are slowed by incongruent global information. We recorded high‐density event‐related potentials (ERPs) to study the role of enhanced local selection in the global precedence effect. Hierarchical stimuli were compound letters in which the local letters were either identical (homogeneous stimuli) or the central local letter was brighter than (bright stimuli) or different in color from the others (red stimuli). Subjects were asked to attend to the pop‐out local letter of the red and bright stimuli during the local task whereas there was no such instruction for the homogeneous stimuli. Top‐down attention to the pop‐out local item weakened the global reaction time advantage and the interference effect. The enhanced local selection decreased the amplitude of an occipito‐temporal negativity between 240–360 msec but increased the amplitude of a frontal‐central negativity between 260–320 msec related to local processing. The incongruency between global and local letters enlarged the posterior N2 in the local condition and this effect was eliminated by enhanced local selection. These effects were evident regardless of whether the pop‐out local letter was defined by color or luminance difference. The results support the proposal that distinct neural mechanisms over the posterior and anterior areas are engaged in the selection process that contributes to local processing of compound stimuli. Hum. Brain Mapping 19:273–281, 2003. © 2003 Wiley‐Liss, Inc.
The Zero‐to‐Three diagnostic classification: A contribution to the validation of this classification from a sample of 85 under‐threes
Authors: Guédeney Nicole , Guédeney Antoine , Rabouam Catherine , Mintz Anne‐Sophie , Danon Gisèle , Moralès Huet Martine , Jacquemain Frédérique
Published: July 2003

The goal of this article is to contribute to the validity of the Zero‐to‐Three diagnostic classification (DC: 0–3) by studying the range, the indices of consistency, the interjudge reliability, and the issue of comorbidity on Axis I. Eighty‐five consecutive children under three years of age and their families were assessed in six mental health centers with clinical interviews and developmental testing. Diagnoses on the five axes of the classification system were made after clinical consensus. The Task Force data record DC: 0–3 was completed for each subject. The use of the classification on a sample of 85 children showed good consistency between the different axes, moderate interjudge reliability, and a high percentage of associated diagnoses on Axis I. The DC: 0–3 appears to help the clinician catch the complexity of the clinical situation in planning the therapeutic strategy. However, the objectivity of the diagnostic criteria must be improved if this system is to be used in research settings. ©2003 Michigan Association for Infant Mental Health.
A five‐year Israeli experience with the DC: 0–3 classification system
Authors: Keren Miri , Feldman Ruth , Tyano Sam
Published: July 2003

Clinical and research experience with the DC: 0–3 classification in an Israeli infant mental health unit is presented in this article. The clinical use of the DC: 0–3 diagnoses is described in the context of our routine assessment process and basic theoretical approach. Reasons for referral and distribution of DC: 0–3 diagnoses of 414 infants and parents, assessed at the unit between the years 1996 and 2000, are shown. Twenty‐five percent had no diagnosis on any of the four axes, 29.5% of the infants had both a Primary and a Relational diagnosis, and only 5.6% had a diagnosis on each of the four axes. Less than half (45%) of the infants received a Primary diagnosis, and 52% had a Relational diagnosis. Possible explanations are given, and problems we faced with the use of the classification are described. Research use of the DC: 0–3 classification is illustrated through the summary of a previously published study on diagnoses and play and feeding interactions of referred and nonreferred infants. These research findings had significant implications for our clinical work, such as the validation of the training we gave community nurses, and the usefulness of the routine combined use of categorical and dimensional tools in a clinical setting. ©2003 Michigan Association for Infant Mental Health.
Diagnostic classification: Results from a clinical experience of three years with DC: 0–3
Authors: Cordeiro Maria José , Da Silva Pedro Caldeira , Goldschmidt Teresa
Published: July 2003

Infancy and early childhood are characterized by a dynamic and ever changing process. Since the beginning of their clinical work at the Infancy Unit, the authors were concerned with individual assessment and the questions about the role played by parents as well as by babies in pathology and intervention. In this article, the authors begin with a description of the path that led them to the selection of DC 0–3 as a diagnostic classification system and how this has been instrumental in helping them to better define infant psychopathology and guide them in treatment orientations. Next, they present the results of the application of Axis I and II of DC: 0–3 in their clinical population in the years 1997, 1998, and 1999. The objectives of this study were to learn more about the distribution of mental disorders in a clinical population up to four years of age. The authors attempted to separate infants at risk for developing psychic disorders from those presenting current psychopathology as well as the possible influence of demographic features on this distribution, to define a target population and design adapted therapeutic measures. The identification of these objectives provides the rationale for the use of a diagnostic tool, like DC: 0–3, which is essential to plan clinical activity, to evaluate therapeutic efficacy, and to develop specific programs. ©2003 Michigan Association for Infant Mental Health.
Diagnostic boundaries between regulatory and multisystem developmental disorders: A clinical study
Authors: Cesari A. , Maestro S. , Cavallaro C. , Chilosi A. , Pecini C. , Pfanner L. , Muratori F.
Published: July 2003

The objective of the study was to define diagnostic boundaries between Regulatory Disorders (RD) and Multisystem Developmental Disorders (MSDD). Two groups of 15 subjects, between 20 and 36 months of age, diagnosed as RD or MSDD, according to DC: 0–3 (1994) criteria, were compared in relationship, linguistic, and behavioral profiles using specific instruments. The results showed that both RD and MSDD children were impaired on all linguistic measures with a higher significant impairment of MSDD for communicative gestures, spontaneous speech, and verbal comprehension. The Child Behavior Checklist (CBCL) showed significant differences for withdrawn, and somatic problems. Only the MSDD group reached the clinical cutoff score in total problems, internalizing and withdrawn behaviors. A discriminate analysis showed that the number of communicative gestures and the scores on withdrawn and somatic problems correctly predicted in 86% of cases. It was concluded that RD and MSDD children show quantitative and qualitative differences in many of the linguistic, behavioral, and relational dimensions that were explored. These dimensions may facilitate the differential diagnosis of these disorders. ©2003 Michigan Association for Infant Mental Health.
The Zero‐to‐Three diagnostic classification in an infant mental health clinic: Its usefulness and challenges
Authors: Maldonado‐Durán Martín , Helmig Linda , Moody Chris , Fonagy Peter , Fulz Jim , Lartigue Teresa , Sauceda‐Garcia J. Manuel , Karacostas Velissarios , Millhuff Charles , Glinka Jill
Published: July 2003

The clinical and practical experiences of a multidisciplinary infant mental health team using the DC: 0–3 Diagnostic Classification system are presented. Using data obtained from clinical in‐depth evaluations of 167 consecutive referred infants, the characteristics and clinical features of this sample of children and parents are examined. The principal issues are children and families' socioeconomic status, number and types of stressors, as well as the symptoms or difficulties exhibited by the infants themselves. A significant proportion of infants come from a deprived socioeconomic background, and many are exposed to violence in the home. The diagnoses given to infants and to the kind of relationship they have with the primary caregiver (Axes I and II) are presented, as well as data as to their physical status (Axis III) and stressors (Axis IV). Many children are diagnosed as having regulatory disturbances, predominantly of the hypersensitive and motorically disorganized type. A significant proportion of mothers exhibited depression. About a third of the babies and primary caregivers had a relationship disorder, the predominant category being the underinvolved type. The implications of these findings are discussed, emphasizing the usefulness and frequency of some categories, while questioning the usefulness of other diagnostic categories. The experience of the group contributes to a dialogue and database regarding the clinical usefulness and applicability of this diagnostic classification system. ©2003 Michigan Association for Infant Mental Health.
Exploring psychopathology in early childhood: PTSD and attachment disorders in DC: 0–3 and DSM‐IV
Authors: Stafford Brian , Zeanah Charles H. , Scheeringa Michael
Published: July 2003

Standard nosologies of psychiatric disorders have made preliminary efforts to accommodate developmental differences in symptom manifestation by young children. Significant gaps remain in the validation of disorders for this age group. The DC: 0–3 represents an alternative nosology for disorders of infancy that attempts to complement existing approaches and assist clinicians and researchers. In this commentary we review research on two disorders of early childhood—Posttraumatic Stress Disorder (PTSD), and Reactive Attachment Disorder (RAD)—to illustrate the promises and problems of the DSM‐IV and DC: 0–3 approach to definition and conceptualization of these disorders. In addition we review what is known about RAD and PTSD with regard to key areas of controversy in early childhood psychopathology: (1) continuous versus categorical conceptualization, (2) developmental considerations, and (3) the relationship context. Recommendations are made for future editions of the DSM‐IV and DC: 0–3, as well as for validity research in early childhood disorders. ©2003 Michigan Association for Infant Mental Health.
DC: 0–3 Assessment Protocol Project: Defining a comprehensive information set to support DC: 0–3 diagnostic formulation
Authors: Weston Donna R. , Thomas Jean M. , Barnard Kathryn E. , Wieder Serena , Clark Roseanne , Carter Alice S. , Fenichel Emily
Published: July 2003

The DC: 0–3 Assessment Protocol Project, an initiative of ZERO‐TO‐THREE's diagnostic classification task force, was undertaken in response to the need for an assessment strategy that would support DC: 0–3 diagnostic formulation for both clinicians and researchers. The Project Workgroup developed and pilot tested an assessment protocol that would: (1) define a sufficiently comprehensive information set to direct and organize observations for clinicians and researchers to systematically assign diagnoses and evaluate interrater agreement with respect to DC: 0–3 and DMS‐IV diagnoses; (2) be flexible enough for use in different settings; and (3) offer potential benefit to participating children and families. The DC: 0–3 Assessment Protocol employs observational strategies, multiple visits, and a multimethod approach to assessment that emphasizes the importance of obtaining information about how a young child's behavior varies across observational contexts and with different caregivers. Based on results of pilot testing the protocol, Workgroup members agreed on the need for futher clarification of the criteria for the diagnoses on Axis I, for differentiating Axis I disorders that reside in the child from Axis II disorders conceptualized as disrupted or disordered relationships, and on the need for refinement of Axis V. Implications for clinical practice, training, and research are discussed. ©2003 Michigan Association for Infant Mental Health.
DC: 0–3 in pediatric liaison work with early eating behavior disorders
Authors: Scheer Peter , Dunitz‐Scheer Marguerite , Schein Alexandra , Wilken Markus
Published: July 2003

This article highlights the use of the DC: 0–3 (Diagnostic Classification, Zero‐to‐Three, 1994) with respect to the special conditions of a hospital pediatric liaison service. The DC: 0–3 system itself has had a positive influence on the structure and quality of diagnostic assessment and the setup of treatment plans in this setting. We describe our experiences with DC: 0–3 in a special group of referred infants with severe eating behavior problems that involved approximately 80% of all liaison referrals and 60% of all outpatient referrals to our pediatric hospital. Most referrals involved requests for weaning infants from long term nasogastric‐(NG) and percutaneous endoscopic gastrostomy‐(PEG) tubes. Four clinical vignettes demonstrate the practical use of DC: 0–3 and its multiple axes in clinical situations. ©2003 Michigan Association for Infant Mental Health.
Oxidative stress during leukocyte absorption apheresis
Authors: Hirayama Aki , Nagase Sohji , Ueda Atsushi , Ishizu Takashi , Taru Yoshinori , Yoh Keigyou , Hirayama Kouichi , Kobayashi Masaki , Koyama Akio
Published: July 2003

Leukocyte absorption apheresis absorbs leukocytes to the apheresis columns involving leukocyte activation. This process is regarded as bioincompatible and avoided in hemodialysis or other extracorporeal circulation processes. Thus, leukocyte apheresis has a potential risk to exacerbate in vivo oxidative stress. We evaluated the changes in plasma oxidative stress during leukocyte apheresis. Patients diagnosed as ulcerative colitis (UC) and treated with leukocyte apheresis were studied. Adacolumn (celluloseacetate beads) or Cellsorba EX (polyethylenephtarate fiber) was used for the leukocyte absorption device. Oxidative stress was measured by thiobarbituric acid reactive substances (TBARS) and hydroxyl radical (•OH) scavenging activity. Plasma samples were collected from the pre‐ and post‐column sampling port at the start, and from the pre‐column sampling port at the end of the treatment. The •OH signal intensities (OHRI) significantly increased during a column passage, indicating a loss of plasma •OH scavenging activity. However, OHRI was reduced at the end, suggesting a recovery of radical scavenging activity during leukocyte apheresis. Significant decreases of OHRI and TBARS were only observed in the early phase of the therapeutic course. No differences of OHRI and TBARS levels were observed between the two columns. These results indicate that though the plasma antioxidant activity was diminished by a column passage, plasma antioxidant activity recovers during the procedure. This efficient antioxidative effect is limited to the early phase of the therapeutic course. J. Clin. Apheresis 18:61–66, 2003. © 2003 Wiley‐Liss, Inc.
Ubiquitin‐dependent and ‐independent proteasomal degradation of apoB associated with endoplasmic reticulum and golgi apparatus, respectively, in HepG2 cells
Authors: Liao Wei , Chang Benny Hung‐Junn , Mancini Michael , Chan Lawrence
Published: July 2003

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B‐100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin‐dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi‐associated apoB was not ubiquitinated, indicating an ubiquitin‐independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER‐associated proteasomal degradation of apoB in the ER is ubiquitin‐dependent whereas that occurring in the Golgi is ubiquitin‐independent. J. Cell. Biochem. 89: 1019–1029, 2003. © 2003 Wiley‐Liss, Inc.
Enhanced binding of Sp1/Sp3 transcription factors mediates the hyperoxia‐induced increased expression of the lung type I cell gene T1α
Authors: Cao Yu‐Xia , Ramirez Maria I. , Williams Mary C.
Published: July 2003

The transcription factor Sp1 plays an important regulatory role in transactivation of the lung type I cell differentiation gene T1α. Like other lung cells, type I cells may encounter changes in oxygen concentration during the lifetime of the organism. We found that exposure of mice to hyperoxia rapidly increases expression of T1α and other type I cell genes, and that returning the mice to normoxia quickly decreases expression. Likewise hyperoxia increases both endogenous T1α expression in lung epithelial cell lines and transcription of luciferase (Luc) from T1α promoter deletion constructs. Using wild‐type promoter fragments and gel shift assays, we determined that Sp1/Sp3 and a key Sp cis‐element in the proximal promoter mediate the hyperoxic response. Mutations of this element and inhibition of Sp–DNA binding by mithramycin block the hyperoxic response. Western analyses of cell homogenates show that the overall abundance of Sp1 and Sp3 proteins is not altered by hyperoxia. However, the abundance of nuclear Sp1 increases after short hyperoxic exposures, suggesting that signaling pathways activated by hyperoxia lead to Sp protein translocation, perhaps as a result of increased Sp phosphorylation. J. Cell. Biochem. 89: 887–901, 2003. © 2003 Wiley‐Liss, Inc.
NF‐κB activation plays an antiapoptotic role in human leukemic K562 cells exposed to ionizing radiation
Authors: Cataldi Amelia , Rapino Monica , Centurione Lucia , Sabatini Nadia , Grifone Giovanna , Garaci Francesco , Rana Rosalba
Published: July 2003

Exposure of cells to ionizing radiation (IR) determines cellular lesions, such as DNA and membrane damage, which involve a coordinate network of signal transduction pathways responsible for resistance to or delay of apoptosis, depending on cell type and administered dose. Since, after IR exposure, the apoptotic profile appeared different in the two chosen cell lines K562 and Jurkat along with caspase‐3 activation, we paid attention to the influence exerted by Protein kinase C δ on transcription factor NF‐κB activation. Interestingly, K562 resist to IR carrying out a survival strategy which includes PKC δ/NF‐κB pathway activation, probably mediated by novel IKKs and a role for PI‐3‐kinase in activating PKC δ at Thr 505 by PDK‐1 phosphorylation is suggested. In addition, since caspase‐3 is not activated in these cells upon ionizing radiation exposure, it could be supposed that NF‐κB antagonizes apoptosis induction interfering with pathways which lead to caspase activation, may be by inducing expression of IAP, caspases 3, 7, 9, inhibitor. Thus NF‐κB activation explains the resistance displayed by K562 to IR and drug potential interference directed to this protein could overcome apoptosis resistance in clinical settings. J. Cell. Biochem. 89: 956–963, 2003. © 2003 Wiley‐Liss, Inc.
Constitutive NO synthase regulates the Na+/Ca2+ exchanger in human T cells: Role of [Ca2+]i and tyrosine phosphorylation
Authors: Kiang Juliann G. , McClain David E. , Warke Vishal G. , Krishnan Sandeep , Tsokos George C.
Published: July 2003

For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+]i) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95‐mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L‐NIL (an iNOs inhibitor). Heat stress increased [Ca2+]i in Jurkat T cells by decreasing the Km of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+]i and a decrease in [Na+]i greater than in cells heated without LNNA, a larger decrease in Km of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress. J. Cell. Biochem. 89: 1030–1043, 2003. Published 2003 Wiley‐Liss, Inc.
Requirement of intracellular free thiols for hydrogen peroxide‐induced hypertrophy in cardiomyocytes
Authors: Tanaka Hiroyuki , Sakurai Koichi , Takahashi Kiyoshi , Fujimoto Yukio
Published: July 2003

Reactive oxygen species (ROS) are by‐products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H2O2 produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H2O2 on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H2O2 induced hypertrophy in control cells in a time‐dependent manner. Pre‐incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H2O2 as compared to non‐BSO pre‐incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre‐incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H2O2, although total thiol levels were not markedly decreased. In the cells pre‐incubated with BSO, hypertrophy was not observed by the addition of H2O2 at any level of concentration. N‐acetyl‐l‐cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H2O2 but also induced hypertrophy in cells pre‐incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H2O2. On the other hand, the hypertrophied cells did not become larger by adding H2O2, but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress. J. Cell. Biochem. 89: 944–955, 2003. © 2003 Wiley‐Liss, Inc.
Paralemnin of the lens
Authors: Bagchi M. , Katar M. , Lo W.‐K. , Maisel H.
Published: July 2003

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X‐100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes. J. Cell. Biochem. 89: 917–921, 2003. © 2003 Wiley‐Liss, Inc.
1α, 25‐Dihydroxy‐vitamin D3 alters syk activation through FcγRII in monocytic THP‐1 cells
Authors: Agramonte‐Hevia José , Hallal Claudia , Garay‐Canales Claudia , Guerra‐Araiza Christian , Camacho‐Arroyo Ignacio , Ortega Soto Enrique
Published: July 2003

In monocytes and macrophages, activation of the tyrosine kinase Syk is an essential step in the biochemical cascade linking aggregation of receptors for immunoglobulin G (FcγR) to initiation of effector functions. An increase in Syk activation during differentiation of myeloid cells by different agents has been reported. We studied the activation state of Syk in response to FcγRII crosslinking in monocytic cells before and after in vitro differentiation with 1α, 25‐dihydroxy‐vitamin D3. We show here that while in undifferentiated THP‐1 cells clustering of FcγRII induces significant phosphorylation and activation of Syk, in THP‐1 cells differentiated in vitro by 1α, 25‐dihydroxy‐vitamin D3, FcγRII crosslinking induced a decrease in Syk activity. In vitro differentiation did not induce changes in the expression of FcγRII isoforms. The observed effect on Syk activation though FcγRII could be mediated by differentiation‐induced changes in the expression and basal activation level of Syk, as well as changes in the association of Syk with the tyrosine phosphatase SHP‐1. These results suggest that the biochemical signaling pathways induced by FcγRII could be dependent on the differentiation state of the cell. J. Cell. Biochem. 89: 1056–1076, 2003. © 2003 Wiley‐Liss, Inc.
Proteomics in biomarker discovery and drug development
Authors: He Qing‐Yu , Chiu Jen‐Fu
Published: July 2003

Proteomics is a research field aiming to characterize molecular and cellular dynamics in protein expression and function on a global level. The introduction of proteomics has been greatly broadening our view and accelerating our path in various medical researches. The most significant advantage of proteomics is its ability to examine a whole proteome or sub‐proteome in a single experiment so that the protein alterations corresponding to a pathological or biochemical condition at a given time can be considered in an integrated way. Proteomic technology has been extensively used to tackle a wide variety of medical subjects including biomarker discovery and drug development. By complement with other new technique advances in genomics and bioinformatics, proteomics has a great potential to make considerable contribution to biomarker identification and to revolutionize drug development process. This article provides a brief overview of the proteomic technologies and their application in biomarker discovery and drug development. J. Cell. Biochem. 89: 868–886, 2003. © 2003 Wiley‐Liss, Inc.
Co‐detection of PTH/PTHrP receptor and tartrate resistant acid phosphatase in osteoclasts
Authors: Gay Carol V. , Zheng Betty , Gilman Virginia R.
Published: July 2003

Serial sections of rat metaphyses were prepared from paraffin embedded tissue blocks and analyzed in sets of three. The central section was stained for tartrate resistant acid phosphatase (TRAP) in order to identify osteoclasts, one adjacent section was immunostained with an affinity purified antibody to a 15 amino acid sequence unique to rat PTH/PTHrP receptor, and the other adjacent section in the set served as an immunostaining control. This allowed each of the 110 osteoclasts examined to be identified by TRAP and to be tested for the presence or absence of PTH/PTHrP receptor. All antibody solutions and rinses contained 1% donkey serum and 0.5% Tween 20 to ensure antibody integrity and good rinsing procedure. Confocal microscopy was used to evaluate fluorescence intensity of the immunostained osteoclasts. Pixel intensities of 58 osteoclasts from young (4 month) rats and 52 osteoclasts from old (15 month) rats were obtained. Pixel intensities were similar (P = 0.89) for both young and old animals. However, the number of PTH/PTHrP receptor deficient osteoclasts was greater for the older animals (14.29% vs. 7.24%). This provides direct evidence of PTH/PTHrP receptors in osteoclasts. J. Cell. Biochem. 89: 902–908, 2003. © 2003 Wiley‐Liss, Inc.
1α,25‐Dihydroxyvitamin D3‐3β‐(2)‐bromoacetate, an affinity labeling derivative of 1α,25‐dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells
Authors: Swamy Narasimha , Persons Kelly S. , Chen Tai C. , Ray Rahul
Published: July 2003

In this report we describe that 1,25(OH)2D3‐3‐BE, a VDR‐affinity labeling analog of 1,25(OH)2D3, showed strong and dose‐dependent growth‐inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF‐7 breast cancer, PC‐3, and LNCaP prostate cancer and PZ‐HPV‐7 immortalized normal prostate cell‐lines. Furthermore, 10−6 M of 1,25(OH)2D3‐3‐BE induced apoptosis specifically in LNCaP and PC‐3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)2D3‐3‐BE) was not due to probable degradation (hydrolysis) of 1,25(OH)2D3‐3‐BE or random interaction of this molecule with cellular proteins. Tissue‐ or cell‐specific action of 1,25(OH)2D3 and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)2D3 and its analogs in various cell‐lines potentially limits their application as anticancer agents. We showed that 1,25(OH)2D3‐3‐BE displayed similar growth‐inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen‐independent PC‐3 cell‐lines. Therefore, these results raise the possibility that 1,25(OH)2D3‐3‐BE or similar VDR‐cross linking analogs of 1,25(OH)2D3 might be considered for further development as potential candidates for prostate cancer. J. Cell. Biochem. 89: 909–916, 2003. © 2003 Wiley‐Liss, Inc.
Induced subgraphs of prescribed size
Authors: Alon Noga , Krivelevich Michael , Sudakov Benny
Published: July 2003

A subgraph of a graph G is called trivial if it is either a clique or an independent set. Let q(G) denote the maximum number of vertices in a trivial subgraph of G. Motivated by an open problem of Erdős and McKay we show that every graph G on n vertices for which q(G)≤ C log n contains an induced subgraph with exactly y edges, for every y between 0 and nδ (C). Our methods enable us also to show that under much weaker assumption, i.e., q(G) ≤ n/14, G still must contain an induced subgraph with exactly y edges, for every y between 0 and $e^{\Omega} (\sqrt { \log\, n})$. © 2003 Wiley Periodicals, Inc. J Graph Theory 43: 239–251, 2003
Off‐diagonal and asymptotic results on the Ramsey number r(K2,m,K2,n)
Authors: Lortz Roland , Mengersen Ingrid
Published: July 2003

An upper bound on the Ramsey number r(K2,n‐s,K2,n) where s ≥ 2 is presented. Considering certain r(K2,n‐s,K2,n)‐colorings obtained from strongly regular graphs, we additionally prove that this bound matches the exact value of r(K2,n‐s,K2,n) in infinitely many cases if $s \approx 2\sqrt n$ holds. Moreover, the asymptotic behavior of r(K2,m,K2,n) is studied for n being sufficiently large depending on m. We conclude with a table of all known Ramsey numbers r(K2,m,K2,n) where m,n ≤ 10. © 2003 Wiley Periodicals, Inc. J Graph Theory 43: 252–268, 2003
A 2‐factor with two components of a graph satisfying the Chvátal‐Erdös condition
Authors: Kaneko Atsushi , Yoshimoto Kiyoshi
Published: July 2003

Chvátal and Erdös showed that a k‐connected graph with independence number at most k and order at least three is hamiltonian. In this paper, we show that a graph contains a 2‐factor with two components, i.e., the graph can be divided into two cycles if the graph is k(≥ 4)‐connected with order at least six and independence number at most k. © 2003 Wiley Periodicals, Inc. J Graph Theory 43: 269–279, 2003
Decreasing the diameter of cycles
Authors: Grigorescu Elena
Published: July 2003

Alon, Gyárfás, and Ruszinkó 1 established a bound for the minimum number of edges that have to be added to a graph of n ≥ n0 vertices in order to obtain a graph of diameter 2. Alon et al. approximated n0 by a polynomial function of the maximum degree of the initial graph. They conjectured that the minimum value for n0 is 12 for the case of Cn, as opposed to n0 = 274 obtained by calculations for the general case. In this paper we prove their conjecture. For the reduction to diameter 3 of a cycle, we also improve the bounds from 1 showing that the minimum number of edges that need to be added to the cycle Cn is between n − 59 and n − 8. © 2003 Wiley Periodicals, Inc. J Graph Theory 43: 299–303, 2003
Tumor necrosis factor‐α‐induced cell death in U373 cells overexpressing α‐synuclein
Authors: Stefanova Nadia , Schanda Kathrin , Klimaschewski Lars , Poewe Werner , Wenning Gregor K. , Reindl Markus
Published: July 2003

Intracellular α‐synuclein inclusion formation in glial cells is frequently seen in Parkinson's disease and multiple system atrophy. Microglial activation in these neurodegenerative disorders suggests that neuroinflammatory responses might interact with α‐synuclein and contribute to the pathogenesis of these disorders. To study the role of tumor necrosis factor‐α (TNF‐α), an important proinflammatory cytokine produced by microglia, on cells overexpressing α‐synuclein we have used the astrocytoma cell line U373 engineered to express C‐terminally truncated α‐synuclein as a fusion protein with red or green fluorescent proteins. We demonstrate that α‐synuclein overexpression augmented TNF‐α‐induced apoptotic cell death in U373 cells by induction of caspase activation. Furthermore, TNF‐α exposure was associated with significant cytoskeletal changes characterized by altered inclusion composition with loss of cytoskeletal proteins and elevation of high‐molecular‐weight α‐synuclein species. We conclude that α‐synuclein overexpression significantly increases the vulnerability of U373 cells to apoptosis through TNF‐α‐mediated pathways. © 2003 Wiley‐Liss, Inc.
Effect of salt stress on crotonobetaine and D(+)‐carnitine biotransformation into L(−)‐carnitine by resting cells of Escherichia coli
Authors: Cánovas Manuel , Torroglosa Tomás , Kleber Hans‐Peter , Iborra Jose Luis
Published: July 2003

The biotransformation of crotonobetaine and D(+)‐carnitine into L(−)‐carnitine is affected by salt stress in the resting cells of E. coli O44 K74 and the transformed E. coli K38 pT7‐5KE32. A yield of 65 and 80% of L(−)‐carnitine, respectively, were obtained with 0.5 M NaCl with the wild and transformed strain compared with the 40% obtained with the control. Higher salt levels reduced the conversion. In L(−)‐carnitine transport studies using both strains, the transformed strain presented slightly lower apparent Km and V values. Arsenate reduced both the transport and biotransformation of crotono‐betaine in the presence or absence of 0.5 M NaCl, whereas vanadate only inhibited these processes under salt stress conditions. Hg(II) inhibited both the transport and biotransformation and Pb(II) reduced the biotransformation only under salt stress conditions. Cu(II) produced a significantly higher decrease than Pb(II) in the biotransformation with both substrates in the absence of salt stress condi‐tions, but only affected transport in the presence of such conditions. Furthermore, salt stress affected the CaiT transporter for L(−)‐carnitine and crotonobetaine and induced ProU and ProP in the absence of the inducer of the L(−)‐carnitine metabolism. It is highly likely that the increase in L(−)‐carnitine production was not only due to improved transport but also to the permeabilization effect caused by NaCl, as transport and 1‐N‐phenylnaphthylamine uptake studies revealed.
Xylanolytic complex from Aspergillus giganteus: production and characterization
Authors: Coelho Glauciane Danusa , Carmona Eleonora Cano
Published: July 2003

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 °C and under shaking at 120 rpm during 84h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar‐cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 °C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T50 of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 °C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg+2, Cu+2 and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin‐layer chromatography indicated endoxylanase activity, lack of debranching enzymes and β‐xylosidase activity in assay conditions.
Purification and characterization of the main laccase produced by the white‐rot fungus Pleurotus pulmonarius on wheat bran solid state medium
Authors: Marques de Souza Cristina Giatti , Peralta Rosane Marina
Published: July 2003

The wood‐degrading fungus Pleurotus pulmonarius produces at least two laccase isoforms, Lcc1 and Lcc2, when grown on wheat bran solid state medium. The main laccase, Lcc2, was purified to apparent electrophoretic homogeneity by using acetone precipitation, anion‐exchange chromatography and gel filtration. Lcc2 had been purified 5.9‐fold with a yield of 49%. A specific activity of 19,750 U/mg protein was found using syringaldazine as a substrate under standard assay conditions. The enzyme is a homodimeric glycoprotein containing 44% glycosilation and an apparent molecular mass of 46 kDa. Type I and type III Cu2+ centers were identified by spectrophotometry. The laccase showed optimal activity at pH 6.2–6.5, 4.0–5.5, and 6.0–8.0 with syringaldazine, ABTS and guaiacol as substrates, respectively. For all substrates, the highest oxidation rates were obtained at 50 °C. The enzyme was stable over a large range of pH (4.5–8.0) and at temperatures up to 50 °C. Under standard assay conditions, the apparent KM values were 12, 210 and 550 μM for syringaldazine, ABTS and guaiacol, respectively. Purified Lcc2 was strongly inhibited by sodium azide, 2‐mercaptoethanol and Hg2+, and slightly inhibited by Mn+2 and the chelant agents, EDTA and EGTA. The enzyme was activated by Cu2+ and it retained a high percentage of its activity in the presence of organic solvents, such as acetonitrile and acetone.
Does the detoxification of penicillin side‐chain precursors depend on microsomal monooxygenase and glutathione S‐transferase in Penicillium chrysogenum?
Authors: Emri Tamás , Oláh Brigitta , Sámi László , Pócsi István
Published: July 2003

The glutathione (GSH) S‐conjugation of 1,2‐epoxy‐3‐(4′‐nitrophenoxy)propane was catalysed predominantly by microsomal glutathione S‐transferase (mGST) in Penicillium chrysogenum. The specific mGST activity unlike the cytosolic GST (cGST) activity increased substantially when the penicillin side‐chain precursor phenoxyacetic acid (POA) was included in the culture medium. Therefore, a microsomal monooxygenase (causing possible release of epoxide intermediates) and mGST‐dependent detoxification pathway may exist for the side‐chain precursors as an alternative to microsomal activation to acyl‐CoA and subsequent transfer to β‐lactam molecules. The P. chrysogenum pahA and Aspergillus nidulans phacA gene products, which are cytochrome P450 monooxygenases and are able to hydroxylate phenylacetic acid (PA) at position 2 on the aromatic ring, are unlikely to release toxic epoxide intermediates but epoxidation of PA and POA due to the action of other microsomal monooxygenases cannot be excluded. The GSH‐dependent detoxification of POA was provoked by a well‐controlled transient lowering of pH (down to 5.0) at the beginning of the production phase in a fed‐batch fermentation system. Both the specific GST and γGT activities were increased but the intracellular GSH concentrations remained unaltered unless the pH of the feed was transiently lowered below 5.0. At pH 4.6, the GSH pool was depleted rapidly but no antibiotic production was observed. Although sucrose was taken up effectively by the cells, cell death and autolysis were progressing. Therefore, the industrial exploitation of the GSH‐dependent detoxification of penicillin side‐chain precursors to reduce intracellular GSH‐levels in order to avoid the GSH inhibition of the β‐lactam biosynthetic enzymes seems to be rather unlikely. P. chrysogenum mGST and cGST were separated using GSH‐Sepharose 6B affinity chromatography. The purified cGST possessed a homodimer (α2) tertiary structure with Mr, α = 29500.