bluuebunny/crossref_metadata_2025 huggingface dataset:
Asymmetry of subinsular anisotropy by in vivo diffusion tensor imaging
Authors: Cao Yue , Whalen Stephen , Huang Jie , Berger Kevin L. , DeLano Mark C.
Published: September 2003

The cortical regions specialized in speech–language exhibit a left–right asymmetry, e.g., a larger cortical size in the left auditory cortex and Wernicke's area. The possibility of developmental asymmetry in axonal fibers interconnecting speech–language cortical areas can be investigated by in vivo diffusion tensor imaging. Fifteen right‐handed native English speakers showed a markedly significant asymmetry (P < 0. 0005) in the relative anisotropy of water diffusion in the subinsular white matter, greater on the left. Additionally, the first principal diffusivity was greater and the second and third principal diffusivities were smaller on the left than right side. These results suggest the subinsular axonal structures developed differently between the left and right sides. A possible association between the hemispheric specialization in language and speech and the subinsular axonal fiber development is discussed. Hum. Brain Mapp. 20:82–90, 2003. © 2003 Wiley‐Liss, Inc.
Attenuation of brain BOLD response following lipid ingestion
Authors: Noseworthy Michael D. , Alfonsi Jeff , Bells Sonya
Published: September 2003

A great deal of heterogeneity exists in fMRI data. Even within the same subject, results on successive days or scan sessions often differ in the number of significantly activated pixels and/or the intensity of activation. We sought to assess whether controllable physiologic modulators, such as dietary factors, could influence the outcome of fMRI data. A high fat diet, for example, prior to a fMRI scan could change microvascular blood rheologic factors and potentially alter brain blood oxygen‐level dependent (BOLD) signal patterns. In healthy adult volunteers, we measured brain BOLD signal during bilateral finger tapping (2 Hz) in the fasted state, and at 40 and 100 minutes post‐ingestion of a 235 mL can of Ensure Plus (Ross Labs), alone or supplemented with either 25cc or 50cc of canola oil. Both the 25cc and 50cc Canola oil treatments produced a significant bilateral decrease in BOLD signal 40 and 100 minutes postprandial. No significant effect was observed with Ensure in the absence of oil. Therefore, to decrease fMRI within and between subject heterogeneity, and thereby increase fMRI statistical power, it is suggested that scanning within 2 hours post high fat ingestion should be avoided. As a corollary, a thorough understanding of a subject's physiological state, prior to an fMRI exam, may reduce the impact of other confounding variables. Hum. Brain Mapp. 20:116–121, 2003. © 2003 Wiley‐Liss, Inc.
Tonotopic cortical representation of periodic complex sounds
Authors: Cansino Selene , Ducorps Antoine , Ragot Richard
Published: September 2003

Most of the sounds that are biologically relevant are complex periodic sounds, i.e., they are made up of harmonics, whose frequencies are integer multiples of a fundamental frequency (Fo). The Fo of a complex sound can be varied by modifying its periodicity frequency; these variations are perceived as the pitch of the voice or as the note of a musical instrument. The center frequency (CF) of peaks occurring in the audio spectrum also carries information, which is essential, for instance, in vowel recognition. The aim of the present study was to establish whether the generators underlying the 100m are tonotopically organized based on the Fo or CF of complex sounds. Auditory evoked neuromagnetic fields were recorded with a whole‐head magnetoencephalography (MEG) system while 14 subjects listened to 9 different sounds (3 Fo × 3 CF) presented in random order. Equivalent current dipole (ECD) sources for the 100m component show an orderly progression along the y‐axis for both hemispheres, with higher CFs represented more medially. In the right hemisphere, sources for higher CFs were more posterior, while in the left hemisphere they were more inferior. ECD orientation also varied as a function of the sound CF. These results show that the spectral content CF of the complex sounds employed here predominates, at the latency of the 100m component, over a concurrent mapping of their periodic frequency Fo. The effect was observed both on dipole placement and dipole orientation. Hum. Brain Mapping 20:71–81, 2003. © 2003 Wiley‐Liss, Inc.
The Faculty Makeover: What Does It Mean for Students?
Authors: Schuster Jack H.
Published: September 2003

Contingent faculty members spend a greater proportion of their overall time teaching, but the preliminary evidence suggests that these appointees are less accessible to students, bring less scholarly authority to their jobs, and are less integrated into the campus culture.
Changing Relationships, Changing Values in the American Classroom
Authors: Townsend Robert B.
Published: September 2003

Although administrators describe the use of adjunct faculty as allowing their institutions to teach more for less, it has instead become a way to earn more in tuition revenue while providing less in education.
Part‐Time Faculty: Why Should We Care?
Authors: Murphy Nutting Maureen
Published: September 2003

Even at institutions where highly trained, highly motivated, and deeply committed part‐timers teach, part‐time instruction can adversely affect teaching, the support of students, the general quality of education, and morale in departments.
Contingent Faculty and Student Learning: Welcome to the Strativersity
Authors: Thompson Karen
Published: September 2003

If we deconstruct the university—and set aside the trendy rhetoric of a multiversity—are we not actually approaching a “strativersity,” a hierarchy of teaching that imposes a hierarchy of learning?
The Choices Before Us: An Administrator's Perspective on Faculty Staffing and Student Learning in General Education Courses
Authors: Reichard Gary W.
Published: September 2003

The only real leverage available to secure the resources needed to improve compensation and other conditions for part‐time, contingent faculty will be our ability to demonstrate persuasively that student learning outcomes are improved as a result.
A Regional Accreditation Perspective on Contingent Faculty Appointments
Authors: Elman Sandra E.
Published: September 2003

When the time that contingent faculty spend grading papers, advising students, and attending department meetings is uncompensated, or when curricular development and academic program review are viewed as ancillary activities, then the vitality and integrity of the teaching‐learning process are compromised.
Reappraisal and Implications for Policy and Research
Authors: Benjamin Ernst
Published: September 2003

We need to recognize and to convince policymakers that excessive dependence on contingent appointments is detrimental to undergraduate learning, and especially so for the “at‐risk” students, unable to attend the few selective institutions that still staff their core programs with full‐time, fully supported faculty.
Progression through the G1‐phase of the on‐going cell cycle
Authors: Boonstra Johannes
Published: September 2003

Cell cycle progression is dependent upon the action of cyclins and their partners the cyclin dependent kinases (CDKs). Each cell cycle phase has its own characteristic cyclin‐CDK combination, cyclin D‐CDK4,6 and cyclin E‐CDK2 being responsible for progression through G1‐phase into S‐phase. Progression through G1‐phase is regulated by signal transduction cascades activated by polypeptide growth factors and by extracellular matrix (ECM) components. Studies aiming to unravel the molecular mechanism by which these extracellular components activate the cyclin‐CDK complexes in the G1‐phase, are usually performed using serum‐starved cells (G0 cells). These cells are activated by addition of growth factors, or the cells are detached from the substratum by trypsinization and subsequently allowed to re‐attach. An alternative approach, however, is to study the effects of growth factors and attachment in the ongoing cell cycle by synchronization of the cells by the mitotic shake‐off method. These cells are not serum starved and not actively detached from the substratum. In this contribution it is shown that both methods yield significant different results. These observations demonstrate that data obtained with model systems should be interpreted with care, especially if the findings are used to explain cell cycle progression in cells in an intact organism. J. Cell. Biochem. 90: 244–252, 2003. © 2003 Wiley‐Liss, Inc.
Catalase, but not MnSOD, inhibits glucose deprivation‐activated ASK1‐MEK‐MAPK signal transduction pathway and prevents relocalization of Daxx: Hydrogen peroxide as a major second messenger of metabolic oxidative stress
Authors: Song Jae J. , Lee Yong J.
Published: September 2003

Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation‐induced cytotoxicity and c‐Jun N‐terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU‐145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal‐regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H2O2) rather than superoxide anion (O2•−) acts as a second messenger of metabolic oxidative stress to activate the ASK1‐MAPK/extracellular signal‐regulated kinase (ERK) kinase (MEK)‐mitogen‐activated protein kinase (MAPK) signal transduction pathway. J. Cell. Biochem. 90: 304–314, 2003. © 2003 Wiley‐Liss, Inc.
Effects of long‐term administration of N‐3 polyunsaturated fatty acids (PUFA) and selective estrogen receptor modulator (SERM) derivatives in ovariectomized (OVX) mice
Authors: Zeitlin L. , Segev E. , Fried A. , Wientroub S.
Published: September 2003

We studied the beneficial effects of dietary consumption of n‐3 polyunsaturated fatty acids (PUFA) and two selective estrogen receptor modulator (SERM) derivatives (SERM‐I and SERM‐II) and their combined effect on serum lipids, skin dermis and adipose layers, bone marrow adipogenesis, and cytokine secretion in mice. Two different ovariectomized (OVX) models were studied: treatment began immediately post‐OVX in one and 3 months post‐OVX in the other. Our results showed that n‐3 PUFA and both SERMs decreased triglyceride levels in the serum, and that SERMs also decreased serum cholesterol levels while n‐3 PUFA had no similar effect. SERMs had no effect on IL‐6, IL‐1 beta, or IL‐10 levels, but they decreased ex vivo tumor necrosis factor (TNF‐α). N‐3 PUFA decreased secretion of non‐induced IL‐6 and TNF‐α from cultured BMC and IL‐1 beta levels in vivo (i.e., in bone marrow plasma), but its main effect was a significant elevation in the secretion of IL‐10, a known anti‐inflammatory cytokine. OVX‐induced B‐lymphopoiesis was not affected by LY‐139481 (SERM‐I) while LY‐353381 (SERM‐II) exhibited an estrogen‐antagonistic effect in sham and OVX mice and elevated the amount of B‐cells in bone marrow. Fish oil consumption prevented the elevation in B‐lymphopoiesis caused by OVX, but had no curative effect on established augmented B‐lymphopoiesis. This activity could be mediated via the elevation of IL‐10 which was shown to suppress B‐lymphopoiesis. Both SERMs and n‐3 PUFA inhibited the increase in adipose tissue thickness caused by OVX in mice. Our results showed that n‐3 PUFA, could prevent some of the deleterious outcomes of estrogen deficiency that were not affected by SERMs. We observed no significant beneficial effects of the combined administration of SERM‐I, SERM‐II, and PUFA on the studied parameters.The exact mechanism by which polyunsaturated fatty acids exert their activities is still not clear, but peroxisome proliferator‐activated receptors (PPARs) might be involved in processes which are modulated by n‐3 PUFA. J. Cell. Biochem. 90: 347–360, 2003. © 2003 Wiley‐Liss, Inc.
Inorganic phosphate as a signaling molecule in osteoblast differentiation
Authors: Beck George R.
Published: September 2003

The spatial and temporal coordination of the many events required for osteogenic cells to create a mineralized matrix are only partially understood. The complexity of this process, and the nature of the final product, demand that these cells have mechanisms to carefully monitor events in the extracellular environment and have the ability to respond through cellular and molecular changes. The generation of inorganic phosphate during the process of differentiation may be one such signal. In addition to the requirement of inorganic phosphate as a component of hydroxyapatite mineral, Ca10(PO4)6(OH)2, a number of studies have also suggested it is required in the events preceding mineralization. However, contrasting results, physiological relevance, and the lack of a clear mechanism(s) have created some debate as to the significance of elevated phosphate in the differentiation process. More recently, a number of studies have begun to shed light on possible cellular and molecular consequences of elevated intracellular inorganic phosphate. These results suggest a model in which the generation of inorganic phosphate during osteoblast differentiation may in and of itself represent a signal capable of facilitating the temporal coordination of expression and regulation of multiple factors necessary for mineralization. The regulation of protein function and gene expression by elevated inorganic phosphate during osteoblast differentiation may represent a mechanism by which mineralizing cells monitor and respond to the changing extracellular environment. J. Cell. Biochem. 90: 234–243, 2003. Published 2003 Wiley‐Liss, Inc.
In vivo administration of 1,25‐dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH
Authors: Ueno Yutaka , Shinki Toshimasa , Nagai Yumiko , Murayama Hisashi , Fujii Katsuyuki , Suda Tatsuo
Published: September 2003

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose‐dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of the receptor activator of nuclear factor‐κB (NF‐κB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)2D3 was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 μg/kg of 1,25(OH)2D3 inhibited the PTH‐induced RANKL mRNA expression, but 0.5 μg/kg of the vitamin did not inhibit it. Regulator of G protein signaling‐2 (RGS‐2) gene expression was suppressed by 1,25(OH)2D3 dose‐dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)2D3 suppressed PTH‐induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)2D3 stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH‐induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor‐mediated signaling. J. Cell. Biochem. 90: 267–277, 2003. © 2003 Wiley‐Liss, Inc.
A proteomic study of the arabidopsis nuclear matrix
Authors: Calikowski Tomasz T. , Meulia Tea , Meier Iris
Published: September 2003

The eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA‐based chromatin and the RNA‐dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Here, we describe the isolation of the nuclear matrix from the model plant Arabidopsis, its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry. Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two‐dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry after resolution by 1D SDS–PAGE. Among the identified proteins were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56, Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF‐1, HSP/HSC70, and DnaJ, which have also been identified in the nucleolus or nuclear matrix of human cells, as well as a number of novel proteins with unknown function. This study is the first proteomic approach towards the characterization of a higher plant nuclear matrix. It demonstrates the striking similarities both in structure and protein composition of the operationally defined nuclear matrix across kingdoms whose unicellular ancestors have separated more than one billion years ago. J. Cell. Biochem. 90: 361–378, 2003. © 2003 Wiley‐Liss, Inc.
NMDA glutamate receptors are expressed by osteoclast precursors and involved in the regulation of osteoclastogenesis
Authors: Merle Blandine , Itzstein Cecile , Delmas Pierre D. , Chenu Chantal
Published: September 2003

We previously identified functional N‐methyl‐d‐aspartate (NMDA) glutamate receptors in mature osteoclasts and demonstrated that they are involved in bone resorption in vitro. In the present work, we studied the expression of NMDA receptors (NMDAR) by osteoclast precursors and their role in osteoclastogenesis using two in vitro models, the murine myelomonocytic RAW 264.7 cell line and mouse bone marrow cells, both of which differentiate into osteoclasts in the presence of macrophage colony‐stimulating factor (M‐CSF) and Rank ligand (RankL). Using RT‐PCR analysis with specific probes, we showed that RAW 264.7 cells and mouse bone marrow cells express mRNA of NMDAR subunits NMDA receptor 1 (NR1) and NMDA receptor 2 (NR2) A, B, and D. These subunits are expressed all along the differentiation sequence from undifferentiated precursors to mature resorbing osteoclasts. Semi‐quantitative PCR analysis showed no regulation of the expression of these subunits during the differentiation process. Two specific non competitive antagonists of NMDAR, MK801 and DEP, dose‐dependently inhibited osteoclast formation in both models, indicating that osteoclastogenesis requires the activation of NMDAR expressed by osteoclast precursors. MK801 had no effect when added only during the first 2 days of culture, suggesting that NMDAR are rather involved in the late stages of osteoclast formation. Finally, we demonstrated using Western‐blotting and immunofluorescence that activation of NMDAR in RAW 264.7 cells by specific agonists induces nuclear translocation of NF‐kappa B, a factor required for osteoclast formation. Altogether, our results indicate that osteoclast precursors express NMDAR that are involved in the osteoclast differentiation process through activation of the NF‐kappa B pathway. J. Cell. Biochem. 90: 424–436, 2003. © 2003 Wiley‐Liss, Inc.
Effects of iron deficiency on the secretion of interleukin‐10 by mitogen‐activated and non‐activated murine spleen cells
Authors: Kuvibidila Solo , Yu L. , Ode D. , Velez M. , Gardner R. , Warrier R.P.
Published: September 2003

Interleukin (IL)‐10 plays crucial regulatory roles in immune responses by inhibiting the secretion of several cytokines (IL‐2, IL‐12, interferon‐gamma (IFN‐γ)) and lymphocyte proliferation. Iron deficiency, a public health problem for children, alters these immune responses. To determine whether these changes are related to altered IL‐10 secretion, we measured IL‐10 in 24 and 48 h supernatant of spleen cell cultures from iron deficient (ID), control (C), pairfed (PF), and ID mice fed the control diet (iron repletion) for 3 (R3) and 14 (R14) days (d, n = 12/group). Mean levels of hemoglobin, hematocrit, and liver iron stores varied as follows: C ≈ PF ≈ R14 > R3 > ID (P < 0.01). Mean baseline IL‐10 levels of ID mice tended to be higher than those of other groups (P > 0.05, ANOVA). Mean IL‐10 levels secreted by concanavalin A (Con A) and antibody raised against cluster of differentiation molecule 3 (anti‐CD3)‐treated cells (±background) were lower in ID than in C (48 h) and iron replete mice (P < 0.05). Underfeeding also reduced IL‐10 secretion by anti‐CD3‐treated cells (48 h, P < 0.05). Lymphocyte proliferative responses to anti‐CD3 ± anti‐CD28 antibodies were lower in ID than in C and PF mice, and they were corrected by iron repletion (P < 0.05). IL‐10 levels negatively correlated with indicators of iron status (r ≤ −0.285) and lymphocyte proliferation (r ≤ −0.379 [r ≤ −0.743 for ID mice]), but positively correlated with IFN‐γ levels (r ≤ 0.47; P < 0.05). Data suggest that iron deficiency has a generalized deleterious effect on cells that secrete both cytokines. Reduced IL‐10 secretion by activated cells does not overcome the inhibition of lymphocyte proliferation due to other factors of T cell activation that are regulated by iron. J. Cell. Biochem. 90: 278–286, 2003. © 2003 Wiley‐Liss, Inc.
Suppression of AP‐1 constitutive activity interferes with polyomavirus MT antigen transformation ability
Authors: Winnischofer Sheila Maria Brochado , de Oliveira Maria Leonor Sarno , Sogayar Mari Cleide
Published: September 2003

Polyomavirus (Py) encodes a potent oncogene, the middle T antigen (MT), that induces cell transformation by binding to and activating several cytoplasmic proteins which take part in transduction of growth factors‐induced mitogenic signal to the nucleus. We have previously reported that the AP‐1 transcriptional complex is a target for MT during cell transformation although, its activation was not sufficient for establishment of the transformed phenotype. Here we show that expression of a dominant‐negative cJun mutant in MT transformed cell lines inhibits its transformation ability, indicating that constitutive AP‐1 activity is necessary for cell transformation mediated by MT. Evidences also suggest that proliferation of MT transformed cells in low serum concentrations and their ability to form colonies in agarose are controlled by distinct mechanisms. J. Cell. Biochem. 90: 253–266, 2003. © 2003 Wiley‐Liss, Inc.
Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1)
Authors: Zhou Lubing , Cryan Ellen V. , D'Andrea Michael R. , Belkowski Stanley , Conway Bruce R. , Demarest Keith T.
Published: September 2003

We have quantitatively measured gene expression for the sodium‐dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/μg total RNA) relative to that in the kidney (3E + 4 molecules/μg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/μg total RNA), approximately 10‐fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)‐labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/μg total RNA (in skeletal muscle) to 3.2E + 6 molecules/μg total RNA (in kidney), levels 10–100‐fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti‐diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart. J. Cell. Biochem. 90: 339–346, 2003. © 2003 Wiley‐Liss, Inc.
Estrogen receptor isoform‐specific regulation of endogenous gene expression in human osteoblastic cell lines expressing either ERα or ERβ
Authors: Monroe David G. , Getz Barbara J. , Johnsen Steven A. , Riggs B. Lawrence , Khosla Sundeep , Spelsberg Thomas C.
Published: September 2003

Estrogen (17β‐estradiol, E2) plays pivotal roles in the function and maintenance of the skeleton, including the bone‐forming osteoblasts (OBs). The functions of E2 are largely mediated through two distinct estrogen receptor isoforms, ERα and ERβ, both of which are expressed in OBs. The level of each isoform dominates at early or late stages of OB differentiation. To date, only a limited comparison between the transcriptional targets of ERα and ERβ on endogenous gene expression has been reported. We have developed new stable cell lines, which contain doxycycline (Dox)‐inducible ERα and ERβ, in the U2OS human osteosarcoma to determine the global transcriptional profile of ERα‐ and ERβ‐regulation of endogenous gene expression. The U2OS‐ERα and U2OS‐ERβ cell lines were treated with Dox and either vehicle control or E2 for 24 h. Gene expression analysis was performed using a microarray containing ∼6,800 full‐length genes. We detected 63 genes that were regulated solely by ERα and 59 genes that were only regulated solely by ERβ. Of the ERα‐regulated genes, 81% were upregulated and 19% were inhibited. Similarly 76% of the ERβ‐regulated genes were upregulated whereas 24% were inhibited by E2. Surprisingly, only 17 genes were induced by both ERα and ERβ. Real‐time PCR was employed to confirm the expression of a selected number of genes. The regulation of a number of known E2‐responsive genes in human OBs, as well as many interesting novel genes, is shown. The distinct patterns of E2‐dependent gene regulation in the U2OS cells by ERα and ERβ shown here suggest that during OB differentiation, when either isoform dominates, a unique pattern of gene responses will occur, partially due to the differentiation state and the ER isoform present. J. Cell. Biochem. 90: 315–326, 2003. © 2003 Wiley‐Liss, Inc.
Hemin‐dependent induction and internalization of CD38 in K562 cells
Authors: Yalcintepe Leman , Ercelen Sebnem , Adin‐Cinar Suzan , Badur Selim , Tiryaki Demir , Bermek Engin
Published: September 2003

The cell surface antigen, CD38, is a bifunctional ecto‐enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP‐ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time‐dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD+ glycohydrolase and ADP‐ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP‐ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti‐CD38 monoclonal antibody. SDS–PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin‐dependent appearence of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin‐induced expression of CD38 was followed by its down‐regulation. J. Cell. Biochem. 90: 379–386, 2003. © 2003 Wiley‐Liss, Inc.
Curcumin inhibits UV irradiation‐induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells
Authors: Chan Wen‐Hsiung , Wu Chih‐Ching , Yu Jau‐Song
Published: September 2003

Ultraviolet (UV) light is a strong apoptotic trigger that induces caspase‐dependent biochemical changes in cells. Previously we showed that UV irradiation can activate caspase‐3, and the subsequent cleavage and activation of p21Cdc42/Rac‐activated kinase 2 (PAK2) in human epidermoid carcinoma A431 cells. In this study we demonstrate that curcumin (Cur), the yellow pigment of Curcuma longa with known anti‐oxidant and anti‐inflammatory properties, can prevent UV irradiation‐induced apoptotic changes, including c‐Jun N‐terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP), mitochondrial release of cytochrome C, caspase‐3 activation, and cleavage/activation of PAK2 in A431 cells. Flow cytometric analysis using the cell permeable dye 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) as an indicator of reactive oxygen species (ROS) generation revealed that the increase in intracellular oxidative stress caused by UV irradiation could be abolished by Cur. In addition, we found that SP600125, a JNK‐specific inhibitor, reduced UV irradiation‐induced JNK activation as well as caspase‐3 activation, indicating that JNK activity is required for UV irradiation‐induced caspase activation. Collectively, our results demonstrate that Cur significantly attenuates UV irradiation‐induced ROS formation, and suggest that ROS triggers JNK activation, which in turn causes MMP change, cytochrome C release, caspase activation, and subsequent apoptotic biochemical changes. J. Cell. Biochem. 90: 327–338, 2003. © 2003 Wiley‐Liss, Inc.
Direct, rapid effects of 25‐hydroxyvitamin D3 on isolated intestinal cells
Authors: Phadnis Ruta , Nemere Ilka
Published: September 2003

Scattered reports in the literature have suggested that the metabolite 25‐hydroxyvitamin D3 [25(OH)D3] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D3 resulted in enhanced transport of 45Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D3 concentrations on 45Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7‐week old chicks were treated either with 25, 100, or 300 nM 25(OH)D3, or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D3 resulted in a significant (P < 0.05) reduction in 45Ca levels, relative to controls, between 1–10 min after treatment, while 300 nM 25(OH)D3 resulted in a significant increase in 45Ca levels, relative to controls, after 10 min of incubation. The effect of 100 nM 25(OH)D3 (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25‐dihydroxyvitamin D3. In cell preparations from 14‐ or 28‐week old birds 100nM 25(OH)D3 had no effect, relative to vehicle controls. Incubation of cells with 2 μM BAY K8644, a calcium channel activator, stimulated 45Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 μM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls). Finally, cells were treated with 100 nM 25(OH)D3 or vehicle and samples taken at various times for analyses of protein kinase C and A activities. No effect of 25(OH)D3 on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D3 addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions. We conclude that 25(OH)D3 has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway. J. Cell. Biochem. 90: 287–293, 2003. © 2003 Wiley‐Liss, Inc.
Regionalization of DNA and protein synthesis in developing stages of the parasitic platyhelminth Echinococcus granulosus
Authors: Galindo M. , Paredes R. , Marchant C. , Miño V. , Galanti N.
Published: September 2003

Echinococcus granulosus is a parasitic platyhelminth, which causes cystid hydatid disease, a major zoonosis involving canids as definitive hosts, and both human and herbivorous domestic animals as intermediate hosts. The disease is caused in intermediate hosts by hydatid cysts, formed upon ingestion of E. granulosus eggs excreted by canids. Protoscoleces, the developmental forms of the parasite infective to canids, are formed in the germinal cellular layer of hydatid cysts. We have found that protoscoleces develop from patches of proliferating cells present in the germinal layer of the hydatid cyst, while most of the other cells of the germinal layer are in a resting state. Further, patches of proliferating cells form buds, which elongate and develop a separate population of cycling cells. In these elongated buds, cell differentiation leads to the main structures of the protoscolex. Protein synthesis is very active among cells of early buds and coincides with their proliferating activity. By contrast, protein synthesis presents a much lower activity in the resting cells of the germinal layer surrounding the growing protoscoleces. In elongated buds at different stages of development, protein synthesis is found mainly close to cellular territories in which cell differentiation occurs. In free infective protoscoleces, cells in DNA synthesis are concentrated in the body of the larva while protein synthesis occurs in the entire larva. This is the first description of the regionalization of DNA and protein synthesis in developing stages of E. granulosus. J. Cell. Biochem. 90: 294–303, 2003. © 2003 Wiley‐Liss, Inc.
Role of MAPK phosphorylation in cytoprotection by pro‐vitamin C against oxidative stress‐induced injuries in cultured cardiomyoblasts and perfused rat heart
Authors: Eguchi Masahiro , Monden Kazuaki , Miwa Nobuhiko
Published: September 2003

The reactive oxygen species (ROS) are known to be generated upon post‐ischemic reperfusion (I/R) of the heart, and to injure cardiac muscle cells. The hydrogen peroxide‐induced mortality of rat cardiomyoblasts H2c9 was markedly inhibited by previous administration with auto‐oxidation‐resistant pro‐vitamin C, the 2‐O‐phosphorylated derivative (Asc2P) of ascorbic acid (Asc). The cytoprotection was partially counteracted by an inhibitor of MAPK (mitogen‐activated protein kinase) kinase (MEK) as shown by DNA strand cleavage assay and mitochondrial dehydrogenase assay. Immunostains indicated that phosphorylated MAPK increased in the hydrogen peroxide‐treated cardiomyoblasts, and that this action was moderately inhibited by Asc2P and restored nearly to the initial, pretreatment level by combined administration of the MEK inhibitor and Asc2P. The I/R‐induced cell injuries in perfused rat hearts as estimated by extracellular release of the cardiac enzyme CPK were inhibited by 2‐O‐α‐glucosylascorbic acid (Asc2G) and Asc, whereas the observed cytoprotection for the cardiomyoblasts was partially counteracted by the MEK inhibitor. The increase in phosphorylated MAPK in I/R‐operated hearts was moderately inhibited by pro‐vitamin C, but restored nearly to the normal non‐operated level by combined administration with the MEK inhibitor. This is in contrast to no alteration in levels of non‐phosphorylated MAPK for all the cases examined as shown by Western blots, consistent with results of immunostains for the cardiomyoblasts. The inhibitory effect of the MEK inhibitor on MAPK phosphorylation was, therefore, suggested to counteract the cytoprotective effects of pro‐vitamin C via a thorough interruption of the phosphorylated MAPK signaling pathway. This was not true of ROS‐related events; the scavenging effects of Asc2G and Asc on hydroxyl radicals generated from I/R‐operated heart were not affected by combined administration with the MEK inhibitor, as shown by the spin‐trapping DMPO‐based ESR method. J. Cell. Biochem. 90: 219–226, 2003. © 2003 Wiley‐Liss, Inc.
Fibroblast growth factor receptor‐1 mediates the inhibition of endothelial cell proliferation and the promotion of skeletal myoblast differentiation by SPARC: A role for protein kinase A
Authors: Motamed Kouros , Blake David J. , Angello John C. , Allen Benjamin L. , Rapraeger Alan C. , Hauschka Stephen D. , Sage E. Helene
Published: September 2003

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF‐2) or interfere with complex formation between FGF‐2 and its high‐affinity FGF receptor‐1 (FGFR1), yet both native SPARC and a peptide derived from the C‐terminal high‐affinity Ca2+‐binding region of protein significantly inhibit ligand‐induced autophosphorylation of FGFR1 (>80%), activation of mitogen‐activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF‐2 (>80%). We also report that in the presence of FGF‐2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca2+‐binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)‐deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1‐mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell‐surface, low‐affinity FGF‐2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand‐binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf‐MAPK pathway. SPARC thus modulates the mitogenic effect of FGF‐2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade. J. Cell. Biochem. 90: 408–423, 2003. © 2003 Wiley‐Liss, Inc.
Expression of procollagen C‐proteinase enhancer in cultured rat heart fibroblasts: Evidence for co‐regulation with type I collagen
Authors: Shalitin Noa , Schlesinger Hadassa , Levy Maurice J. , Kessler Efrat , Kessler‐Icekson Gania
Published: September 2003

Procollagen processing by procollagen C‐proteinase (PCP) is an important step in collagen deposition. This reaction is stimulated by another glycoprotein, known as PCP enhancer. The objective of this study was to identify factors that regulate the expression of PCP enhancer in cardiac fibroblasts and examine possible correlation with collagen expression. Rat heart fibroblasts were cultured in the presence or absence of three known stimulators of collagen synthesis: ascorbic acid, TGF‐β, and aldosterone. The mRNA and protein levels of PCP enhancer and collagen type I were each assessed using Northern and Western blotting, respectively. Expression of PCP was assessed by RT‐PCR and its activity in the culture media was determined using radioactive procollagen as the substrate. The levels of PCP enhancer mRNA increased 1.5‐ to 2‐fold in response to ascorbate, TGF‐β, or aldosterone. This increase was paralleled by an up to fourfold increase in the level of the pro α1(I) collagen chain transcript and was accompanied by a marked increase in the levels of the respective proteins in the culture media. PCP activity in the culture media was also increased, apparently, without effect on its expression. These results indicate that expression of PCP enhancer in cultured rat heart fibroblasts is coordinated with that of collagen. The observed augmentation of PCP activity may be a consequence of the increase in the levels of PCP enhancer in the culture media. J. Cell. Biochem. 90: 397–407, 2003. © 2003 Wiley‐Liss, Inc.
Downregulation of inducible nitric oxide synthetase by neurotrophin‐3 in microglia
Authors: Tzeng Shun‐Fen , Huang Hsin‐Ying
Published: September 2003

Microglia activated after many neurological degeneration of the central nervous system (CNS) act as important regulators for neuropathogenesis in the injured CNS via producing proinflammatory mediators, such as nitric oxide (NO), TNF‐α, and IL‐1β. Neurotrophin‐3 (NT‐3) is a well‐known trophic factor for neural survival, development, and plasticity. Activated microglia are NT‐3‐producing cells in the injured CNS, and express its receptor‐TrkC. However, little is known about the effect of NT‐3 on activated microglia. In this study, pre‐treatment of a mouse microglial cell line, BV2, with NT‐3 for 24 h indicated that NT‐3 reduced the inducible form of NO synthase (iNOS), NO, and TNF‐α in BV2 stimulated with lipopolysaccharide (LPS). NT‐3 exerted less effect on the reduction of these proinflammatory mediators when it was added to BV2 cultures either simultaneously with LPS or post LPS treatment. These findings indicate that NT‐3 may serve as an anti‐inflammatory factor to suppress microglial activation. J. Cell. Biochem. 90: 227–233, 2003. © 2003 Wiley‐Liss, Inc.
Unusual atmospheric pressure chemical ionization conditions for detection of organic peroxides
Authors: Rondeau David , Vogel René , Tabet Jean‐Claude
Published: September 2003

Organic peroxides such as the cumene hydroperoxide I (Mr = 152 u), the di‐tert‐butyl peroxide II (Mr = 146 u) and the tert‐butyl peroxybenzoate III (Mr = 194 u) were analyzed by atmospheric pressure chemical ionization mass spectrometry using a water–methanol mixture as solvent with a low flow‐rate of mobile phase and unusual conditions of the source temperature (≤50 °C) and probe temperature (70–200 °C). The mass spectra of these compounds show the formation of (i) an [M + H]+ ion (m/z 153) for the hydroperoxide I, (ii) a stable adduct [M + CH3OH2]+ ion (m/z 179) for the dialkyl peroxide II and (iii) several protonated adduct species such as protonated molecules (m/z 195) and different protonated adduct ions (m/z 227, 389 and 421) for the peroxyester III. Tandem mass spectrometric experiments, exact mass measurements and theoretical calculations were performed for characterize these gas‐phase ionic species. Using the double‐well energy potential model illustrating a gas‐phase bimolecular reaction, three important factors are taken into account to propose a qualitative interpretation of peroxide behavior toward the CH3OH2+, i.e. thermochemical parameters ($\Delta H_{\rm reaction}^{\circ}$) and two kinetic factors such as the capture constant of the initial stable ion–dipole and the magnitude of the rate constant of proton transfer reaction into the loose proton bond cluster. Copyright © 2003 John Wiley & Sons, Ltd.
Electron ionization mass spectrometric study of monomeric models of O‐polysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba
Authors: Kováčik Vladimír , Pätoprstý Vladimír , Oksman Pentti , Mistrík Robert , Kováč Pavol
Published: September 2003

Fragmentation mechanisms of electron ionization (EI) mass spectrometry of the title compounds have been elucidated by high‐resolution (HR) mass spectrometric measurements of the elemental composition and measurements of the metastable transitions (B2/E, CID). The experimental results were interpreted with the help of Mass Frontier 3.0 software, which aided the elucidation of fragmentation mechanisms and helped to deduce structures of the ions formed. Characteristic under the conditions of EI‐MS measurement was the production of protonated adducts. Three distinct pathways observed include the formation of oxonium type ions, the conjugated transfer of electrons in the pyranose ring, and cleavage of the acylamide side chains. By applying the results obtained, the molecular mass, as well as the structures of both the saccharide and acylamide side chain involved in related substances, can be determined. Copyright © 2003 John Wiley & Sons, Ltd.
Role of opposite charges in protein electrospray ionization mass spectrometry
Authors: Šamalikova Maria , Grandori Rita
Published: September 2003

The conformation dependence of protein spectra recorded by electrospray ionization mass spectrometry (ESI‐MS) is an interesting and useful phenomenon, whose origin is still the object of debate. Different mechanisms have been invoked in the attempt to explain the lower charge state of folded versus unfolded protein ions in ESI‐MS, such as electrostatic repulsions, solvent accessibility, charge availability, and native‐like interactions. In this work we try to subject to direct experimental test the hypothesis that conformation‐dependent neutralization of charges with polarity opposite to the net charge of the protein ion could play a critical role in such an effect. We present results of time‐of‐flight nano‐ESI‐MS on the peptide angiotensin II, indicating that negative charges of carboxylate groups can contribute to spectra recorded in positive‐ion mode when stabilized by favorable electrostatic interactions, which is the central assumption of our hypothesis. Comparison of horse and spermwhale myoglobin (Mb) shows that changing the total number of basic residues within a given three‐dimensional structure shifts the charge‐state distribution (CSD) of the folded protein in positive‐ion mode. This result appears to be in contrast to models in which electrostatic repulsions or availability of charges in the ESI droplets represent the limiting factor for the ionization of folded protein ions in ESI‐MS. At the same time, it suggests a role of acidic residues in conformational effects in positive‐ion mode. Furthermore, an attempt is made to rationalize those cases in which, in contrast, the main charge state observed in ESI‐MS under non‐denaturing conditions deviates considerably from the net charge expected on the basis of the amino‐acid composition. These cases usually correspond to proteins with quite balanced content in basic and acidic residues, suggesting that this might be a factor influencing their charging behavior in ESI‐MS. Experiments on mutants of ribonuclease Sa (RNase Sa) reveal that progressively reducing the excess of acidic residues, replacing them by lysine, causes almost no shift in the spectrum of the folded protein in negative‐ion mode. Analogously, variants with an excess of three or five basic residues give similar spectra in positive‐ion mode. These results indicate a lower limit to the extent of ionization observable by ESI‐MS (6− or 8+ in the case of RNase Sa in water). Below such limit of net charge, changes in the relative amount of ionizable side chains do not affect the qualitative features of the observed CSDs. A progressive loss of signal intensity caused by the mutations in negative‐ion mode suggests that low charge states might also be counterselected, even within the m/z range theoretically accessible to the instrument. Copyright © 2003 John Wiley & Sons, Ltd.