bluuebunny/crossref_metadata_2025 huggingface dataset:
Anisodamine causes acyl chain interdigitation in phosphatidylglycerol
Authors: Wang Ping-yuan , Chen Jian-wen , Hwang Fen
Published: July 2002

The effect of anisodamine on the structure of the gel phase and the properties of the acyl chain disordering transition of dipalmitoylphosphatidylglycerol (DPPG) has been studied through high‐sensitivity differential scanning calorimetry (DSC) and fluorescence polarization measurements of 16‐(9‐anthroyloxyl)‐palmitic acid (16AP) and 3‐(9‐anthroyloxyl)‐stearic acid (3AS), labeling, respectively, the ends and the third carbon of the acyl chains. The non‐interdigitated DPPG multilamellar vesicles formed in HEPES buffer show clear fluidity gradient in their acyl chains, whereas the fluidity gradients are completely abolished in the presence of anisodamine. The DSC results showed that the phase transition temperature (T
m) of DPPG is decreased and the enthalpy (ΔH) is increased by anisodamine, while the pre‐transition vanishes. At 3 mM anisodamine, the ΔH of DPPG reaches 9.6 kcal/mol. It can be concluded that DPPG forms an interdigitated gel phase in the presence of anisodamine. A molecular scheme for the interaction of anisodamine with DPPG is proposed.
Differential adrenergic regulation of C/EBPα and C/EBPβ in brown adipose tissue
Authors: Rehnmark Stefan , Antonson Per , Xanthopoulos Kleanthis G. , Jacobsson Anders
Published: July 2002

We investigated the regulation of the expression of two members of the C/EBP family of transcriptional activators, C/EBPα and C/EBPβ, in brown adipose tissue in mice. Less than one hour of cold exposure led to dramatic changes in the expression of both genes. C/EBPα steady‐state mRNA and protein levels were drastically and rapidly reduced whereas C/EBPβ mRNA and protein levels were induced severalfold. Also norepinephrine injection affected the expression of the transcription factors. Preconfluent cells in brown fat primary cultures responded to norepinephrine with a decrease in C/EBPα and an increase in C/EBPβ mRNA; in confluent cells the expression of both factors was increased. Thus, C/EBPα and C/EBPβ gene expression is under adrenergic control both in vivo and in vitro but the type of response is directed by the degree of differentiation of the cells.
Formation of disulphide bonds in the reaction of SH group‐containing amino acids with trimethylamine N‐oxide
Authors: Brzezinski Bogumil , Zundel Georg
Published: July 2002

Two amino acids containing SH group (cysteine and homocysteine)+trimethylamine N‐oxide systems were studied by FTIR and 1H NMR spectroscopy. This study demonstrates that cysteine and homocysteine ethylesters react with trimethylamine N‐oxide. Immediately after mixing, SH⋯ON ⇋ S−⋯H+ ON hydrogen bonds with large proton polarizability are formed. Then a reaction proceeds resulting in the formation of corresponding disulphides. Trimethylamine N‐oxide is present in biological systems. Thus, our results suggest that trimethylamine N‐oxide may play a regulatory role in S‐S bond formation in enzymes and other proteins.
13C NMR study of the mode of interaction in solution of the B fragment of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G
Authors: Kato Koichi , Gouda Hiroaki , Takaha Wakana , Yoshino Atsuko , Matsunaga Chigusa , Arata Yoji
Published: July 2002

The mode of interaction of the B domain (FB) of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G (IgG) has been investigated by 13C NMR spectroscopy. Mouse IgG1, IgG2a, and IgG2b proteins have been selectively labeled with 13C at the carbonyl carbon of His, Met, Trp or Tyr residue and used to prepare the corresponding Fc fragments by limited proteolysis. Site‐specific resonance assignments have been made for each of these Fc analogues. FB was reported to form two contacts (contact 1 and contact 2) with human Fc in the crystal [Biochemistry 20 (1981) 2361‐2370]. Comparisons of the chemical shift data of the Fc fragments observed in the absence and presence of FB have led us to conclude that in solution contact 1 is responsible for the formation of the Fc‐FB complexes.
Kinetics of CO binding to putative Na+‐motive oxidases of the o‐type from Bacillus FTU and of the d‐type from Escherichia coli
Authors: Muntyan M.S. , Bloch D.A. , Drachev L.A. , Skulachev V.P.
Published: July 2002

The kinetics of CO reassociation with isolated Bacillus FTU o‐type oxidase and with solubilized membranes of Escherichia coli (GO 102 strain) containing the d‐type oxidase only, upon laser flash photolysis under reducing conditions, were studied. In both cases, kinetics are shown to be composed of three phases (τ 35–70 μs, 0.25–0.5 ms and 2–5 ms). The spectra of the flash‐induced absorbance changes of the first kinetic components proved to be characteristic of CO‐o‐ and CO‐b595d‐cytochrome complexes in Bac. FTU and E.Coli, respectively. The spectra of the second and the third components appeared to be nearly the same in Bac. FTU and E.Coli with peaks for the former at 436–437 and 590 nm and troughs at 419–420 and 569 nm; and for the latter with peaks at 436–437 and 558–560 nm and troughs at 419–420 and 575–578 nm. The similarity between the putative Na+‐pumping Bac. FTU o‐ and E. Coli d‐type oxidases and their difference from the H+‐motive Bac. FTU caa3− and E. Coli o‐type oxidases are discussed.
Regulation of c‐myc expression by sodium butyrate in the colon carcinoma cell line Caco‐2
Authors: Souleimani Abdellah , Asselin Claude
Published: July 2002

The human colon carcinoma cell line Caco‐2 spontaneously undergoes enterocytic differentiation in culture. We used sodium butyrate to modify differentiation and growth properties of this cell line and considered c‐myc expression as a potential target. Degradation of normal c‐myc mRNAs with a half‐life of 20 min is not coupled to translation in this cell line, as determined by cycloheximide treatment. We show that butyrate reduces c‐myc. mRNA levels after a 30 min delay. Butyrate does not affect c‐myc, expression at the level of transcriptional initiation or elongation, as determined by run‐on analysis, but at a post‐transcriptional level. Cycloheximide blocks butyrate‐dependent reduction of c‐myc mRNA levels. Cross‐linking experiments show that a 34 kDa protein binds specifically to the c‐myc AU‐rich instability determinant found in the 3′‐untranslated region (ARE). Binding of this protein to the ARE is not modulated by butyrate or cycloheximide. These experiments suggest that butyrate induces a factor involved in c‐myc. mRNA degradation that differs from the known ARE‐associated proteins. Post‐transcriptional modification of gene expression could be one of the major targets for this anti‐proliferative agent.
Cleavage of supercoiled double‐stranded DNA by several ribosome‐inactivating proteins in vitro
Authors: Ling Jun , Liu Wang-yi , Wang T.P.
Published: July 2002

Several ribosome‐inactivating proteins (RIPs), such as ricin (including its A‐chain), luffin, cinnamomin and camphorin, were found to express enzymatic activity to cleave supercoiled double‐stranded DNA. In particular, α‐sarcin, a RIP with a novel ribonuclease activity, was first proved to have this activity. They convert supercoiled DNA into a nicked circular conformation at low concentrations and further into a linear form at high concentrations: they have no effect on linear DNA. Although intact type II RIPs exhibited no RNA N‐glucosidase activity, they were detected to cleave supercoiled DNA. Even if ricin A‐chain was treated by boiling, its activity on supercoiled DNA was largely retained.
Sensitivity ofHalobacterium salinariumto attractant light stimuli does not change periodically
Authors: Krohs Ulrich
Published: July 2002

Halobacterium salinariumswims alternately in both directions of its cell axis. The average time between two reversals of the swimming direction is modulated by light stimuli. It is a matter of dispute whether the sensitivity to attractant stimuli depends on the time of stimulation during an interval. This question is crucial for model descriptions of the system. I have confirmed constancy of responsiveness with cells adapted to constant conditions and have reconstructed contradicting results. These are shown to be based on inadequate experimental and evaluative methods. The assumption of self‐sustained oscillations which modulate sensitivity can not be justified from the attractant response.
Involvement of the brain type of ryanodine receptor in T‐cell proliferation
Authors: Hakamata Yasuhiro , Nishimura Seiichiro , Nakai Junichi , Nakashima Yasuyo , Kita Toru , Imoto Keiji
Published: July 2002

Cloning and sequence analysis of cDNA showed that the brain type of ryanodine receptor (RYR) is expressed in human Jurkat T‐lymphocyte cells. Fura‐2 measurements revealed that the RYR in T‐cells functions as a ryanodine‐sensitive, caffeine‐insensitive Ca2+ release channel. Furthermore, ryanodine stimulated proliferation and altered the growth pattern of cultured human T‐cells when added together with FK506.
Yta10p is required for the ATP‐dependent degradation of polypeptides in the inner membrane of mitochondria
Authors: Pajic Alexander , Tauer Reimund , Feldmann Horst , Neupert Walter , Langer Thomas
Published: July 2002

Incompletely synthesized polypeptides in the mitochondrial inner membrane are subject to rapid proteolysis. We demonstrate that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process. Yta10p‐dependent degradation requires divalent metal ions and the hydrolysis of ATP. Yta 10p is an integral protein of the inner mitochondrial membrane exposing the carboxy terminus to the mitochondrial matrix space. Based on the presence of consensus binding sites for ATP, and for divalent metal ions found in a number of metal dependent endopeptidases, a direct role of Yta10p in the proteolytic breakdown of membrane‐associated polypeptides in mitochondria is suggested.
A carotenogenic gene cluster exists on a large plasmid in Thermus thermophilus
Authors: Tabata Kazuyuki , Ishida Sayuri , Nakahara Tadaatsu , Hoshino Takayuki
Published: July 2002

In Thermus thermophilus HB27, the crtB gene encoding phytoene synthase was found to exist on the large plasmid, pTT27. One of the carotenoid under‐producing mutants, Crt31, carried a derivative of pTT27 (pTT27') in which deletion and inversion were observed near the crtB gene. T. thermophilus HB8 also contained a large plasmid which showed homology to pTT27 and the crtB gene. These results suggested that genes for carotenoid biosynthesis occurred as a cluster on a large plasmid in Thermus thermophilus. This is the first report to show directly that carotenogenesis is plasmid‐encoded in microorganisms.
Molecular cloning of rat JAK3, a novel member of the JAK family of protein tyrosine kinases
Authors: Takahashi Takamune , Shirasaw Takuji
Published: July 2002

We have cloned and sequenced a cDNA (JAK3) encoding a novel member of the JAK family of protein tyrosine kinases. JAK3 was identified by RT‐PCR of rat mesangial cells using degenerate oligonucleotide primers, and a full‐length clone was isolated from a rat spleen cDNA library. The primary structure of JAK3 showed cDNA with an open reading frame of 1,100 amino acids which comprises the PTK catalytic domain and a second kinase‐related domain characteristic for JAK kinase. JAK3 was phylogenetically shown to be most closely related to JAK2 among the previously known JAK family members, JAK1, JAK2 and Tyk2. Southern analysis revealed that JAK3 is a single copy gene and well conserved in the vertebral genome. Northern analysis indicated that the 4.0 kb mRNA was transcribed in a variety of tissues including spleen, lung, kidney and intestine.
Low density lipoprotein is saturable by pro‐oxidant copper
Authors: Gieseg Steven P. , Esterbauer Hermann
Published: July 2002

The oxidative resistance of low density lipoprotein (LDL) can be experimentally described by the length of time during which no significant lipid peroxidation is observed in a pro‐oxidant environment. This period of inhibited oxidation, termed the ‘lag phase’, is partially due to the radical scavenging reactions of the anti‐oxidants contained in the LDL particle. We have shown that the LDL lag time decreases with increasing copper concentration, leveling out at a relatively high copper‐to‐LDL ratio. This behaviour demonstrates the existence of a finite number of saturable pro‐oxidant copper binding sites within the LDL particle. The relationship is described by the equation, lag time = [Cu]− · K · t
min + t
min where the constant, K, is the negative reciprocal of the x‐axis intercept of the graphed function, and t
min is given by the y‐axis intercept. By this definition of the constant, K is the amount of copper that will produce a lag time of twice t
min, while t
min is the minimum time a particular LDL will resist oxidation at a maximum copper concentration.
Cerebrovascular amyloidosis in squirrel monkeys and rhesus monkeys: apolipoprotein E genotype
Authors: Morelli Laura , Wei LiHong , Amorim Angela , McDermid Jeanine , Abee Christian R. , Frangione Blas , Walker Lary C. , Levy Efrat
Published: July 2002

Some neuropathological changes characteristic of aging and Alzheimer's disease (AD) in humans are present also in senescent non‐human primates. The human apoE4 allele is associated with an increased risk of developing late‐onset familial and sporadic AD. We found that rhesus monkeys and three subspecies of squirrel monkeys are homozygous for apoE phenotype with arginine at positions 112 and 158 as in human apoE4. However, in both species threonine replaces arginine at position 61 of human apoE. It was previously shown that arginine 61 was critical in determining apoE4 lipoprotein distribution in humans.
Cloning, sequencing and functional assignment of the chlorophyll biosynthesis gene, chlP, of Synechocystis sp. PCC 6803
Authors: Addlesee Hugh A. , Gibson Lucien C.D. , Jensen Poul E. , Hunter C.Neil
Published: July 2002

A gene from the cyanobacterium Synechocystis sp. PCC 6803 has been cloned and sequenced, and subsequently used to partially complement a bchP mutant of the purple photosynthetic bacterium Rhodobacter sphaeroides. This mutant is blocked in the terminal hydrogenation steps of bchla biosynthesis and possesses only bchl esterified with geranylgeraniol. It also has a reduced cellular level of the light‐harvesting LH2 complex, and the 850 nm absorbance maximum of LH2 is red‐shifted by approximately 6 nm. Upon heterologous expression of the Synechocystis bchP homologue, not only are hydrogenated forms of bchlaGG detectable, but the level of LH2 is increased and the red‐shift reversed by several nm. We conclude that this gene, which we term chlP, encodes the enzyme catalysing the stepwise hydrogenation of geranylgeraniol to phytol during chla biosynthesis.
Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor
Authors: Brown Mark T. , McGrath William J. , Toledo Diana L. , Mangel Walter F.
Published: July 2002

The type of proteinase and the nature of the active site of the human adenovirus proteinase are unknown. For these reasons we produced an inhibitor profile of the enzyme. Enzyme activity in disrupted virions was inhibited by several serine‐specific as well as cysteine‐specific proteinase inhibitors. Of the inhibitors that worked, the most useful potentially in illuminating the nature of the active site was bovine pancreatic trypsin inhibitor (BPTI), and for this reason we extensively characterized the interaction with BPTI. In disrupted virions, the enzyme is irreversibly inhibited by BPTI with a K
i of 35 nM and a k
i of 6.2 × 10−4 s−1. One reason enzyme activity is inhibited is that BPTI, a basic protein, precipitates the viral DNA, a cofactor of enzyme activity. In vitro with purified components, BPTI acts as a competitive inhibitor (K
i 2 μM) of the recombinant proteinase complexed with its 11‐amino‐acid cofactor pVIc. The recombinant endoproteinase is heat labile whereas its 11‐amino‐acid cofactor is heat stable. We estimate there are about 50 molecules of proteinase per virus particle.
Determination of the endogenous phosphorylation state of B‐50/GAP‐43 and neurogranin in different brain regions by electrospray mass spectrometry
Authors: Di Luca Monica , Pastorino Lucia , Raverdino Vittorio , De Graan Pierre N.E. , Caputi Antonio , Gispen Willem H. , Cattabeni Flaminio
Published: July 2002

Electrospray mass spectrometry coupled to liquid chromatography was utilized to measure two PKC neuronal substrates, B‐50/GAP‐43 and neurogranin, in single rat brain areas. Aliquots of perchloric acid extracts were directly injected and mass spectra recorded. At elution times of 14.2 and 27.0 min two molecular species of MW 7450 and 23 602 Da were observed. These values are in excellent agreement for the expected MW for rat neurogranin and B‐50/GAP‐43. The presence of molecular species shifted by 80 mass units in both cases indicates that these proteins are present in phosphorylated forms in cortical and hippocampal extracts.
Nitric oxide stimulates stress‐activated protein kinases in glomerular endothelial and mesangial cells
Authors: Pfeilschifter Josef , Huwiler Andrea
Published: July 2002

Exposure of rat glomerular mesangial cells and primary cultures of bovine glomerular endothelial cells to compounds releasing nitric oxide (NO), including MAHMANONOate, S‐nitrosoglutathione, and spermine‐NO, results in a time‐ and concentration‐dependent activation of stress‐activated protein kinases (SAPK) as measured by the phosphorylation of c‐Jun in a solid phase kinase assay. Dibutyryl cGMP had no effect on SAPK activity. Pretreatment of the cells with the tyrosine kinase inhibitor genistein strongly attenuated NO‐induced c‐Jun phosphorylation. Furthermore, N‐acetylcysteine markedly reduced the activation of SAPK in response to NO. These studies identify SAPK as a target for NO which may be critical for the NO‐induced apoptosis of glomerular mesangial and endothelial cells.
Synchronous primary carcinomas of the endometrium and ovary
Authors: Sheu B.‐C. , Lin H.‐H. , Chen C.‐K. , Chao K.‐H. , Shun C.‐T. , Huang S.‐C.
Published: July 2002

Objectives: Synchronous carcinomas of the endometrium and ovary may indicate either independently developing neoplasms or metastatic disease. The clinical implications and prognosis of these two categories are quite different. The objectives of this study were to identify and evaluate the empirical criteria and significant therapeutic implications. Method: The National Taiwan University Hospital Cancer Registry records and pathological reports from 1977 to 1994 were reviewed. Empirical criteria were used to identify synchronous primary cancers. Results: A total of 322 patients had endometrial cancer and 421 patients had ovarian cancer in our Cancer Registry records. Eleven patients had simultaneous cancer involvement of both the endometrium and ovary. Six cases fulfilled the criteria of synchronous primary carcinomas of the endometrium and ovary. Of these, five were alive and free of disease for 35–144 months (median 94.2 months). The disease‐free survival rates between patients with synchronous primary and metastatic cancers of different histologic types showed a statistically significant difference (P = 0.013). No statistical significance was noted for different histologic types (P > 0.5). Conclusions: The empirical criteria used here were useful in identifying synchronous primary cancers of the endometrium and ovary. The favorable clinical outcome may relate to early detection of early‐stage disease and low‐grade malignancy with an indolent growth rate. Surgical management with or without adjuvant therapy has a satisfactory outcome in our experience.
A comparative model of facility network design methodologies
Authors: Powell Robinson E. , Swink Morgan L.
Published: July 2002

Operations managers use a variety of decision making tools when designing facility networks. Intuition, scenario evaluation, heuristic and optimization procedures are commonly applied. This paper discusses the relative advantages and disadvantages of the alternative methodologies for network design and proposes a cost trade‐off model for choosing the best approach. Accessibility, precision and supplemental analysis are key factors to consider during the selection process. We also present the results of a laboratory experiment which measured the performance of analysts using intuition, scenario evaluation and enhanced scenario evaluation procedures to solve realistically‐sized network design problems. The test problems included several sizes of single‐echelon, multi‐activity and two‐echelon facility network design problems. The experimental results, when embedded into the cost trade‐off model, provide quantitative guidelines for selecting the appropriate decision support procedures. Implications for practitioners, researchers and decision support system developers are provided.
MFH‐1 is required for bone morphogenetic protein‐2‐induced osteoblastic differentiation of C2C12 myoblasts
Authors: Yang Xiao-Li , Matsuura Hiroshi , Fu Yan , Sugiyama Toshihiro , Miura Naoyuki
Published: July 2002

Mesenchyme forkhead‐1 (MFH‐1), a winged helix/forkhead transcription factor, is expressed in developing cartilaginous tissues, kidney and arch arteries, and is essential for the normal development of the axial skeleton and aortic arch formation of mice. To investigate the possible role of MFH‐1 in osteogenesis and osteoblast differentiation, we examined expression of MFH‐1 induced by bone morphogenetic protein‐2 (BMP‐2) in C2C12 myoblasts, and found that MFH‐1 protein and also MFH‐1 mRNA increased markedly in C2C12 cells after treatment with BMP‐2. To confirm the hypothesis that BMP‐2 induced osteoblastic differentiation of C2C12 cells by increasing MFH‐1 expression, we lowered the endogenous MFH‐1 level by stably transfecting C2C12 cells with antisense MFH‐1 sequence, and found that in antisense MFH‐1 cell lines, both alkaline phosphatase (ALP) activity and production of osteocalcin induced by BMP‐2 decreased markedly in comparison with control cell lines. Our results suggest that the BMP‐2‐induced MFH‐1 protein may play a key role in regulating the commitment to osteoblastic differentiation of C2C12 myoblasts and production of osteoblast markers including ALP and osteocalcin.
The distribution of T‐DNA in the genomes of transgenic Arabidopsis and rice
Authors: Barakat Abdelali , Gallois Patrick , Raynal Monique , Mestre-Ortega Dominique , Sallaud Christophe , Guiderdoni Emmanuel , Delseny Michel , Bernardi Giorgio
Published: July 2002

Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1–2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene‐rich regions (collectively called the ‘gene space’) separated by vast gene‐empty, repeated sequences. In contrast, in Arabidopsis thaliana, genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T‐DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T‐DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene‐rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.
Mutations in the S4 domain of a pacemaker channel alter its voltage dependence
Authors: Vaca L. , Stieber J. , Zong X. , Ludwig A. , Hofmann F. , Biel M.
Published: July 2002

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization‐activated cyclic nucleotide‐gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately −20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about −20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately −50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.
A peroxidase homologue and novel plastocyanin located by proteomics to the Arabidopsis chloroplast thylakoid lumen
Authors: Kieselbach Thomas , Bystedt Maria , Hynds Peter , Robinson Colin , Schröder Wolfgang P.
Published: July 2002

A study by two‐dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low‐intensity spots and accounting for low‐level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec‐ and delta pH‐dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.
Tenascin‐C induced stimulation of chondrogenesis is dependent on the presence of the C‐terminal fibrinogen‐like globular domain
Authors: Murphy Lyn I. , Fischer Doris , Chiquet-Ehrismann Ruth , Mackie Eleanor J.
Published: July 2002

The relationship between structure of tenascin‐C (Tn‐C), a multi‐domain extracellular matrix protein, and its stimulation of chondrogenesis was examined using recombinant Tn‐C isoforms (full length or with specific domains deleted) as substrata for undifferentiated chicken mesenchymal cells. Of the Tn‐C variants tested, only Tn‐C lacking the fibrinogen‐like domain or Tn‐C comprised solely of fibrinogen‐like domains failed to stimulate chondrogenesis. The ability of variants to stimulate chondrogenesis was not dependent on their ability to support adhesion or stimulate proliferation. These results demonstrate that the fibrinogen‐like domain of Tn‐C is necessary but not sufficient for induction of chondrogenesis.
Inhibition of interleukin‐6 signaling by galiellalactone
Authors: Weidler Marcus , Rether Jan , Anke Timm , Erkel Gerhard
Published: July 2002

A search for inhibitors of the IL‐6‐mediated signal transduction in HepG2 cells using secreted alkaline phosphatase (SEAP) as reporter gene resulted in the isolation of galiellalactone (1) from fermentations of the ascomycete strain A111‐95. Galiellalactone inhibits the IL‐6‐induced SEAP expression with IC50 values of 250–500 nM by blocking the binding of the activated Stat3 dimers to their DNA binding sites without inhibiting the tyrosine and serine phosphorylation of the Stat3 transcription factor. Due to its selective activity, galiellalactone may serve as a lead compound for the development of new therapeutic agents for diseases originating from the inappropriate expression of IL‐6 and as molecular tool to dissect the JAK/STAT pathways.
Peroxisome proliferator‐activated receptor γ activators inhibit interleukin‐12 production in murine dendritic cells
Authors: Faveeuw Christelle , Fougeray Sylvie , Angeli Véronique , Fontaine Josette , Chinetti Giulia , Gosset Philippe , Delerive Philippe , Maliszewski Charlie , Capron Monique , Staels Bart , Moser Muriel , Trottein François
Published: July 2002

Peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear receptor superfamily. They are divided into three subtypes (α, β or δ, and γ) and are involved in lipid and glucose homeostasis and in the control of inflammation. In this study, we analyzed the expression of PPARs in murine dendritic cells (DCs), the most potent antigen presenting cells. We find that immature as well as mature spleen‐derived DCs express PPARγ, but not PPARα, mRNA and protein. We also show that the PPARγ activator rosiglitazone does not interfere with the maturation of DCs in vitro nor modifies their ability to activate naive T lymphocytes in vivo. Finally, we present evidence that PPARγ activators down‐modulate the CD40‐induced secretion of interleukin‐12, a potent Th1‐driving factor. These data suggest a possible role for PPARγ in the regulation of immune responses.
Molecular mechanisms underlying endocytosis and sorting of ErbB receptor tyrosine kinases
Authors: Waterman Hadassa , Yarden Yosef
Published: July 2002

The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand–receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling. Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging. These are the Eps15‐mediated sorting to clathrin‐coated regions of the plasma membrane and the c‐Cbl‐mediated targeting of receptors to lysosomal degradation. Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules. The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described. Finally, we discuss the relevance of receptor down regulation to cancer therapy.
The transcriptional regulator, Arg82, is a hybrid kinase with both monophosphoinositol and diphosphoinositol polyphosphate synthase activity
Authors: Zhang Tong , Caffrey James J , Shears Stephen B
Published: July 2002

The Arg82 gene of Saccharomyces cerevisiae encodes a transcriptional regulator that phosphorylates inositol 1,4,5‐trisphosphate [Saiardi et al. (1999) Curr. Biol. 9, 1323–1326]. However, some controversy has surrounded the nature of the reaction products. We now show that Arg82 phosphorylates inositol 1,3,4,5‐tetrakisphosphate to inositol pentakisphosphate, which is itself converted to two isomers of diphosphoinositol tetrakisphosphate, one of which has never previously been identified. One of the diphosphoinositol phosphates was further phosphorylated by a yeast cell lysate. We propose that Arg82 is an ancestral precursor of two distinct and specific enzyme families: inositol 1,4,5‐trisphosphate kinases and diphosphoinositol polyphosphate synthases.
A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of α‐actinin‐2
Authors: Cukovic D. , Lu G.W-K. , Wible B. , Steele D.F. , Fedida D.
Published: July 2002

The interaction between the amino terminus of Kv1‐type potassium channels and α‐actinin‐2 has been investigated. Using a combination of yeast two‐hybrid analysis and in vitro binding assays, α‐actinin‐2 was found to bind to the N‐termini of both Kv1.4 and Kv1.5 but not to the equivalent segments of Kv1.1, Kv1.2 or Kv1.3. Deletion analysis in the in vitro binding assays delineated the actinin binding region of Kv1.5 to between amino acids 73 and 148 of the channel. The Kv1.5 binding sites in α‐actinin‐2 were found to lie within actinin's internal spectrin repeats. Unlike the reported interaction between actinin and the NMDA receptor, calmodulin was found to have no effect on actinin binding to Kv1.5.
Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle
Authors: Nakamura Masafumi , Zhen Zhou Xiao , Kishi Shuji , Ping Lu Kun
Published: July 2002

Pin2/TRF1 was independently identified as a telomeric DNA‐binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle‐regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule‐disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM‐dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1‐interacting protein, EB1, that was originally identified in our yeast two‐hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co‐localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end‐binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia–telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.
In seedlings of the heavy metal accumulator Brassica juncea Cu2+ differentially affects transcript amounts for γ‐glutamylcysteine synthetase (γ‐ECS) and metallothionein (MT2)
Authors: Schäfer Holger J , Greiner Steffen , Rausch Thomas , Haag-Kerwer Angela
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Enterocyte differentiation is compatible with SV40 large T expression and loss of p53 function in human colonic Caco‐2 cells
Authors: Djelloul Siham , Forgue-Lafitte Marie-Elisabeth , Hermelin Brigitte , Mareel Marc , Bruyneel Erik , Baldi Alfonso , Giordano Antonio , Chastre Eric , Gespach Christian
Published: July 2002

Transfer of the SV40 large‐T (LT) oncogene into isolated human and murine intestinal epithelial cells induced alterations of the ultrastructural organization and polarization of the resulting immortalized cell lines. We now demonstrate that the functional expression of the SV40 LT antigen in Caco‐2 cells did not alter phenotypic markers of differentiation, including expression of villin, sucrase–isomaltase, brush border and dome formation. As compared to parental cells, the transfected Caco‐2 LT9 cells exhibited similar growth curves and no invasive properties in vitro. The major oncogenic function of the SV40 LT antigen in transfected Caco‐2 cells is associated with reduced latency times necessary for the manifestation of tumors in athymic nude mice. The Caco‐2 cell line contained deleted and mutant p53 alleles (stop codon in position 204) and has no detectable truncated p53 protein by Western blot. Molecular complexes between the SV40 LT antigen and the retinoblastoma‐related proteins pRb1 and Rb2 were clearly identified at the different phases of the growth curve. When compared to normal human colonic crypts, Caco‐2 cell differentiation is related to partial redistribution of pRb1 into hypophosphorylated, antiproliferative forms. The pRb2 protein is found elevated in a subset of human colorectal tumors and their corresponding liver metastases. We conclude that: (1) Caco‐2 cells exert a dominant control against the oncogenic functions of the LT antigen; (2) loss of p53 function is not restrictive for the establishment of polarity and differentiation of the enterocyte lineage; (3) the levels and phosphorylation status of the Rb1 and Rb2 proteins may play important roles in the proliferation and differentiation of normal and neoplastic human colonic mucosa.
Phosphorylation of microtubule‐associated protein tau by stress‐activated protein kinases
Authors: Goedert Michel , Hasegawa Masato , Jakes Ross , Lawler Sean , Cuenda Ana , Cohen Philip
Published: July 2002

The paired helical filament, which comprises the major fibrous element of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated microtubule‐associated protein tau. Many of the hyperphosphorylated sites in tau are serine/threonine‐prolines. Here we show that the stress‐activated protein (SAP) kinases SAPK1γ (also called JNK1), SAPK2a (also called p38, RK, CSBPs, Mpk2 and Mxi2), SAPK2b (also called p38β), SAPK3 (also called ERK6 and p38γ) and SAPK4 phosphorylate tau at many serine/threonine‐prolines, as assessed by the generation of the epitopes of phosphorylation‐dependent anti‐tau antibodies. Based on initial rates of phosphorylation, tau was found to be a good substrate for SAPK4 and SAPK3, a reasonable substrate for SAPK2b and a relatively poor substrate for SAPK2a and SAPK1γ. Phosphorylation of tau by SAPK3 and SAPK4 resulted in a marked reduction in its ability to promote microtubule assembly. These findings double the number of candidate protein kinases for the hyperphosphorylation of tau in Alzheimer's disease and other neurodegenerative disorders.
Copper and cell‐oxidized low‐density lipoprotein induces activator protein 1 in fibroblasts, endothelial and smooth muscle cells
Authors: Mazière Cécile , Djavaheri-Mergny Mojgan , Frey-Fressart Véronique , Delattre Jacques , Mazière Jean-Claude
Published: July 2002

The effect of cupric ion‐ or endothelial cell‐oxidized low‐density lipoproteins (LDL) on transcription factor AP1 activation was investigated by electrophoretic mobility shift assay. Both oxidized LDL induced AP1 activation in fibroblasts, endothelial and smooth muscle cells. This phenomenon was also observed in the presence of cycloheximide. α‐Tocopherol, a lipophilic free radical scavenger, and N‐acetylcysteine, an hydrophilic antioxidant, partially inhibited the stimulatory effect of Cu2+‐oxidized LDL. LDL modified by the mixture of the oxygen radicals OH· and O2·−, which generated lipid peroxidation products, also initiated AP1 activation, whereas LDL modified by OH· alone, which did not lead to marked LDL lipid peroxidation, was ineffective. Thus, lipid peroxidation products seem at least partially involved in the activation mechanism. Since AP1 activity is essential for the regulation of genes involved in cell growth and differentiation, our study suggests that the oxidative stress induced by oxidized LDL might be related to the fibroproliferative response observed in the atherosclerotic plaque.
Overexpression of the mouse dishevelled‐1 protein inhibits GSK‐3β‐mediated phosphorylation of tau in transfected mammalian cells
Authors: Wagner Uta , Brownlees Janet , Irving Nicholas G , Lucas Fiona R , Salinas Patricia C , Miller Christopher C.J
Published: July 2002

Tau is a neuronal microtubule‐associated protein whose function is modulated by phosphorylation. GSK‐3β is a tau kinase. GSK‐3β is part of the wingless signalling pathway and stimulation by wingless is predicted to down‐regulate GSK‐3β activity. In Drosophila imaginal disc cells, overexpression of dishevelled, a component of the wingless pathway, mimics the wingless signal. We have therefore studied the effect that overexpression of the murine dishevelled‐1 protein has on GSK‐3β‐mediated phosphorylation of tau in transfected CHO cells. We find that co‐transfection with dishevelled‐1 is inhibitory to GSK‐3β‐mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimer's disease and the possible relevance of these findings to Alzheimer's disease pathogenesis are discussed.
The Arabidopsis Cks1At protein binds the cyclin‐dependent kinases Cdc2aAt and Cdc2bAt
Authors: De Veylder Lieven , Segers Gerda , Glab Nathalie , Casteels Peter , Van Montagu Marc , Inzé Dirk
Published: July 2002

In Arabidopsis, two cyclin‐dependent kinases (CDK), Cdc2aAt and Cdc2bAt, have been described. Here, we have used the yeast two‐hybrid system to identify Arabidopsis proteins interacting with Cdc2aAt. Three different clones were isolated, one of which encodes a Suc1/Cks1 homologue. The functionality of the Arabidopsis Suc1/Cks1 homologue, designed Cks1At, was demonstrated by its ability to rescue the temperature‐sensitive cdc2‐L7 strain of fission yeast at low and intermediate expression levels. In contrast, high cks1At expression levels inhibited cell division in both mutant and wild‐type yeast strains. Cks1At binds both Cdc2aAt and Cdc2bAt in vivo and in vitro. Furthermore, we demonstrate that the fission yeast Suc1 binds Cdc2aAt but only weakly Cdc2bAt, whereas the human CksHs1 associated exclusively with Cdc2aAt.
Laminin α2 chain‐null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)‐deficient congenital muscular dystrophy
Authors: Miyagoe Yuko , Hanaoka Kazunori , Nonaka Ikuya , Hayasaka Michiko , Nabeshima Yoko , Arahata Kiichi , Nabeshima Yo-ichi , Takeda Shin'ichi
Published: July 2002

Using the gene targeting technique, we have generated a new mouse model of congenital muscular dystrophy (CMD), a null mutant for the laminin α2 chain. These homozygous mice, designated dy3K/dy3K
, are characterized by growth retardation and severe muscular dystrophic symptoms and die by 5 weeks of age. Light microscopy revealed that muscle fiber degeneration in these mice begins no later than postnatal day 9. In degenerating muscles, considerable amounts of TUNEL positive nuclei were detected as well as DNA laddering, suggesting increased apoptotic cell death was involved in the process of muscle fiber degeneration.
Transgene expression in Xenopus rods
Authors: Knox Barry E , Schlueter Charisse , Sanger Brooke M , Green Carla B , Besharse Joseph C
Published: July 2002

The photoreceptors of the vertebrate retina express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod‐ and cone‐specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219–226). The mechanisms that regulate gene expression in a rod/cone‐specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99–126, Wiley‐Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173–3183) can be used to study cell‐specific expression in the retina. We have used a 5.5 kb 5′ upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179–3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina and pineal, which is apparent by 4 days post‐nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.
A novel basement membrane‐induced gene identified in the human endometrial adenocarcinoma cell line HEC1B
Authors: Treeck Oliver , Strunck Elisabeth , Vollmer Günter
Published: July 2002

Cultured HEC1B human endometrial adenocarcinoma cells respond to reconstituted basement membrane (Matrigel) by morphological and functional differentiation in vitro. Our goal is to identify genes involved in this differentiation process. By means of rt‐PCR, we were able to isolate the novel 2.4 kb Matrigel‐induced transcript icb‐1 containing an open reading frame predicting a 31.7 kDa protein. The time‐dependent induction of icb‐1 gene expression by basement membrane was confirmed by Northern blot experiments. In a data bank search, several EST homologues corresponding to the 3′ untranslated region could be found. In summary, icb‐1 as a new tool enables us to study molecular mechanisms of cell‐matrix interactions contributing to carcinogenesis.
Zeatin is indispensable for the G2‐M transition in tobacco BY‐2 cells
Authors: Laureys Françoise , Dewitte Walter , Witters Erwin , Van Montagu Marc , Inzé Dirk , Van Onckelen Harry
Published: July 2002

LC‐MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG‐CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well‐defined cytokinin (here, zeatin) in the G2‐M transition of tobacco BY‐2 cells.
Interleukin‐1 receptor accessory protein interacts with the type II interleukin‐1 receptor
Authors: Malinowsky David , Lundkvist Johan , Layé Sophie , Bartfai Tamas
Published: July 2002

Stably transfected HEK‐293 cells express on their surface the murine type II IL‐1 receptor (mIL‐1RII) as demonstrated by FACS analysis using the mAb 4E2, however binding of [125I]‐hrIL‐1β to these cells is nearly absent. Saturable high affinity binding of [125I]‐hrIL‐1β is observed when the murine IL‐1 receptor accessory protein (mIL‐1RAcP) is coexpressed with mIL‐1RII. Binding of [125I]‐hrIL‐1β to mIL‐1RII‐mIL‐1RAcP complex can be inhibited either with antibodies to mIL‐1RII (mAb 4E2), or by antibodies to mIL‐1RAcP (mAb 4C5). The number of high affinity binding sites in cells stably transfected with the cDNA for mIL‐1RII is dependent on the dose of cDNA for mIL‐1RAcP used to transfect the cells. The high affinity complex between mIL‐1RII and mIL‐1RAcP is not preformed by interaction between the intracellular domains of these two transmembrane proteins, rather it appears to require the extracellular portions of mIL‐1RII and mIL‐1RAcP and the presence of a ligand. We suggest that in addition to its earlier described decoy receptor role, IL‐1RII may modulate the responsiveness of cells to IL‐1 by binding the IL‐1RAcP in unproductive/non‐signalling complexes and thus reducing the number of signalling IL‐1RI‐IL‐1RAcP‐agonist complexes when IL‐1 is bound.
Potential dual targeting of an Arabidopsis archaebacterial‐like histidyl‐tRNA synthetase to mitochondria and chloroplasts1
Authors: Akashi Kinya , Grandjean Olivier , Small Ian
Published: July 2002

A cDNA clone encoding a histidyl‐tRNA synthetase (HisRS) was characterized from Arabidopsis thaliana. The deduced amino acid sequence (AtHRS1) is surprisingly more similar to HisRSs from archaebacteria than those from eukaryotes and prokaryotes. AtHRS1 has an N‐terminal extension with features characteristic of mitochondrial and chloroplast transit peptides. Transient expression assays in tobacco protoplasts clearly demonstrated efficient targeting of a fusion peptide consisting of the first 71 amino acids of AtHRS1 joined to jellyfish green fluorescent protein (GFP) to both mitochondria and chloroplasts. These observations suggest that the AtHisRS1 cDNA encodes both mitochondrial and chloroplast histidyl‐tRNA synthetases.