bluuebunny/crossref_metadata_2025 huggingface dataset:
Subunit III of the chloroplast ATP‐synthase can form a Ca2+‐binding site on the lumenal side of the thylakoid membrane
Authors: Zakharov Stanislav D. , Ewy Robert G. , Dilley Richard A.
Published: July 2002

Subunit III, the 8 kDa component of the chloroplast CF0 H+ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca2+‐binding properties. After n‐butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrenedivinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic‐solvent soluble preparation were used in a 45Ca2+‐ligand blot assay known to detect high affinity Ca2+‐binding sites in proteins. Polypeptides were separated with SDS‐PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl2 in the presence of 10‐fold excess of MgCl2 and 200‐fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de‐formylation of the N‐terminal methionine, or with a subsequent treatment with La3+. Ca2+ binding was maximum at pH 7.5–8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivativizes the hydrophobically located Glu‐61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca2+ binding. The data indicate that the carbonyl group of the formylated N‐terminal Met‐1 and probably the carboxyl group of the subunit III C‐terminal Val‐81 provide some of seven essential oxygen ligands normally required for defining a Ca2+‐binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca2+‐binding site. Based on the accepted models for the hairpin conformation of the subunit III, it does seem clear that the Ca2+‐binding site can form on the lumenal side of the membrane in the functional CF0 structure.
Expression of different mitogen‐regulated protein/proliferin mRNAs in Ehrlich carcinoma cells
Authors: Gil-Torregrosa Beatriz , Urdiales José L. , Lozano José , Mates José M. , Sanchez-Jimenez Francisca
Published: July 2002

Results from in vivo and from serum‐free primary cultures of Ehrlich cells suggest that the expression of mitogen‐regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL‐related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF‐1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.
Complete nucleotide sequence and genome organization of a tobamovirus infecting cruciferae plants
Authors: Dorokhov Yu.L. , Ivanov P.A. , Novikov V.K. , Agranovsky A.A. , Morozov S.Yu. , Efimov V.A. , Casper R. , Atabekov J.G.
Published: July 2002

Genomic RNA sequence of a tobamovirus infecting cruciferae plants (cr‐TMV) was determined. The RNA is composed of 6312 nucleotides and contains four ORFs encoding the proteins of 122K (ORF1), 178K (ORF2), 29K (ORF3) and 18K (capsid protein, ORF4). ORF4 overlaps ORF3 by 74 nucleotides and the overlapping region can be folded into a stable hairpin structure. The 3′‐terminal region of the cr‐TMV RNA preceding the tRNA‐like structure was shown to form six potentially stable pseudoknots.
Protein phosphatase 1 can modulate alternative 5′ splice site selection in a HeLa splicing extract
Authors: Cardinali Beatrice , Cohen Patricia T.W. , Lamond Angus I.
Published: July 2002

Recent studies using HeLa in vitro splicing extracts have shown that changes in the relative concentrations of constitutive protein splicing factors can affect the choice between competing 5′ splice sites in alternatively spliced mammalian pre‐mRNAs. Here we report that treatment of a HeLa splicing extract with human protein phosphatase 1 strongly inhibits formation of mRNA spliced to the distal 5′ splice site while stimulating relative use of the proximal 5′ splice site. This effect is not observed if spliceosomes assemble prior to protein phosphatase 1 treatment. These data show that alternative splicing in HeLa extracts can be mediated by changes in protein modification as well as by changes in the relative concentration of splicing factors. Changes in protein phosphorylation may thus provide a rapid mechanism for cells to respond to stimuli that require an alteration in alternative splicing patterns.
Characterisation of the F‐actin binding domains of villin: classification of F‐actin binding proteins into two groups according to their binding sites on actin
Authors: Pope Brian , Way Michael , Matsudaira Paul T. , Weeds Alan
Published: July 2002

The F‐actin binding properties of chicken villin, its headpiece and domains 2–3 (V2‐3) have been analysed to identify sites involved in bundle formation. Headpiece and V2‐3 bind actin with K
d values of ~7 μM and ~0.3 μM, respectively, at low ionic strength. V2‐3 binding, like that of villin, is weakened with increasing salt concentration; headpiece binding is not. Competition experiments show that headpiece and V2‐3 bind to different sites on actin, forming the two cross‐linking sites of villin. Headpiece does not compete with the F‐actin binding domains of gelsolin or α‐actinin, but it dissociates actin depolymerizing factor. We suggest that the F‐actin binding domains of actin severing, crosslinking and capping proteins can be organized into two classes.
Substrate specificity of Escherichia coli LD‐carboxypeptidase on biosynthetically modified muropeptides
Authors: Leguina José Ignacio , Quintela José Carlos , de Pedro Miguel A.
Published: July 2002

Escherichia coli murein can be biosynthetically modified. Amino acids at positions 3 and 4 (m‐diaminopimelic acid and D‐alanine, respectively) on the peptide moieties can be changed under appropriate growth conditions. The activity of E. coli LD‐carboxypeptidase on biosynthetically modified substrates has been studied in vitro. The enzyme hydrolysed all tested disaccharide‐tetrapeptide monomeric muropeptides modified at position 4. Monomers with m‐lanthionine, but not with L‐ornithine, instead of m‐diaminopimelic acid at position 3 were accepted. However, neither cross‐linked muropeptides nor macromolecular murein were substrates for the reaction. Our observations argue against a direct effect of ld‐carboxypeptidase on macromolecular murein metabolism.
Enhanced intracellular delivery of doxorubicin by scavenger receptor‐mediated endocytosis for preferential killing of histiocytic lymphoma cells in culture
Authors: Basu Sunanda , Mukhopadhyay Bratati , Basu Sandip K. , Mukhopadhyay Amitabha
Published: July 2002

A conjugate of the antineoplastic drug doxorubicin (DXR) with maleylated bovine serum albumin (MBSA) was taken up by a human histiocytic lymphoma cell line (U937) through the high efficiency process of scavenger receptor‐mediated endocytosis resulting in a sixfold higher intracellular concentration of the drug compared to that obtained when the free drug was administered. Compared to the free drug, the drug conjugate showed significantly higher cytotoxicity towards U937 cells presumably because of intracellular availability of a pharmacologically active form of the drug. The intracellular product released after lysosomal degradation of the drug conjugate was chromatographically identical to free DXR. These findings merit serious consideration in the development of new chemotherapeutic agents for the treatment of histiocytic malignancies.
Spectroscopic study of an HIV‐1 capsid protein major homology region peptide analog
Authors: Clish Clary B. , Peyton David H. , Barklis Eric
Published: July 2002

The capsid (CA) domain of retroviral Gag proteins posseses one subdomain, the major homology region (MHR), which is conserved among nearly all avian and mammalian retroviruses. While it is known that the mutagenesis of residues in the MHR will impair virus infectivity, the precise structure and function of the MHR is not known. In order to obtain further information on the MHR, we have examined the structure of a synthetic peptide encompassing the MHR of human immunodeficiency virus type I (HIV‐1) CA protein. Multiple sequence alignment and secondary structure prediction indicate that the peptide could form 50% α‐helix and 10% β‐sheet. In addition, circular dichroism studies indicate that, in the presence of 50% trifluoroethanol (TFE), the peptide adopts an α‐helical structure over half of its length. Further analysis by proton nuclear magnetic resonance spectroscopy suggests that the C‐terminal portion of the MHR forms a helix in aqueous solution. Upon the addition of TFE, the position of the helix remains nearly constant, but the magnitude of the changes in Hα chemical shifts of the residues indicate a more stable helix. These results suggest that a helical C‐terminus of retroviral MHRs may be integral to the function of this region.
Changes in fast axonal transport in sensory neurons during tadpole metamorphosis
Authors: Tang H.Z. , Hammerschlag R. , Chan H. , Smith R.S.
Published: July 2002

Fast axonal transport of radiolabeled protein was examined in lumbar and tail dorsal root ganglion (DRG) neurons at progressive stages of bullfrog tadpole metamorphosis. Accumulation of [35S]methionine‐labeled protein proximal to a lumbar peripheral nerve ligature (at a fixed distance from the DRG) increased as tadpoles advanced from premetamorphosis through prometamorphosis to metamorphic climax. The rate of increase was steeper when expressed as a percentage of protein synthesized in the neurons of origin than when expressed as a percentage of total DRG protein synthesis. Further, the increase was not secondary to a rise in protein synthesis. In contrast, fast axonal transport decreased in DRG neurons of the tail at the onset of metamorphic climax, when tail resorption is initiated. The stage‐related increase in protein transport in lumbar nerves is due, at least in part, to an increased rate of transport. As determined from optically detected anterograde organelles in individual lumbar nerve axons, an approximate doubling of the fast transport rate occurred between the premetamorphic stage and metamorphic climax. In addition, the rates of organelle transport in lumbar axons of adult bullfrogs were significantly greater than in corresponding axons of tadpoles at metamorphic climax, further suggesting that organelle velocity is a developmentally regulated parameter of fast axonal transport.
Development of vestibular function: biochemical, morphological and electronystagmographical assessment in the rat
Authors: Meza Graciela , Acuña Dora , Gutiérrez Angel , Merchan Jaime M. , Rueda Joaquín
Published: July 2002

Glutamate decarboxylase and choline acetyltransferase were measured in homogenated ampullar cristae of rats during development from postnatal day 13 to 60 to determine changes in levels of these enzymes during early postnatal development. Afferent and efferent innervation of the hair cells of the developing cristae were studied using electron microscopy. In parallel, groups of rats, postrotatory nystagmus were used to assess the development of semicircular canal function during the same time interval. The level of glutamate decarboxylase was high on postnatal day 15 and did not change notably over the remaining days to day 60. Activity of choline acetyltransferase was nearly absent at day 15, but reached levels seen in mature animals by day 17, and remained almost unchanged thereafter. In contrast, as revealed by electronmicroscopy, afferent and efferent innervation appeared to be mature by day 8. Postrotatory nystagmus presented the adult‐like features from day 19 onward. According to these results, a role for glutamate decarboxylase in afferent transmission is suggested by the parallel development of levels of glutamate decarboxylase and afferent innervation of the ampullary cristae. The finding of a similar time course of development of choline acetyltransferase levels and postrotatory nystagmus suggests that a cholinergic efferent innervation is involved in the onset of vestibular‐ocular function.
Postnatal development of glycosidases and gangliosides in the rat central nervous system
Authors: Prasad Vidudala V.T S.
Published: July 2002

The developmental profiles of sialidase, β‐galactosidase, β‐hexosaminidase and β‐glucosidase were compared to those of the gangliosides in rat brain and spinal cord. The glucosidase activities (enzyme units/g wet tissue), except β‐galactosidases, were found to be higher in brain than spinal cord, in adult rats. Among the hydrolases, β‐hexosaminidase showed a higher level of activity in both brain and spinal cord. In brain, the hydrolases, except β‐glucosidase, followed a similar developmental pattern, showing an increase from birth to 21 days, and then decreased to adult values by day 90. In the spinal cord, sialidase, β‐galactosidase, pH 3.1, and β‐hexosaminidase activities increased from birth to 21 days, reaching peak values. These activities then declined to adult values by 90 days of age. However, β‐galactosidase, pH 4.5, and β‐glucosidase activities showed a peak at day 14. Brain total ganglioside concentration (μg N‐acetylneuraminic acid/g tissue) increased slowly between birth and 7 days of age, followed by a rapid phase of increase to attain a peak value by day 21. The concentration of total gangliosides in the spinal cord is less when compared to the brain. The proportions of individual gangliosides in the central nervous system also varied during development. The rapid phase of increase in enzyme activities between 0–7 and 14–21 days and a decrease thereafter is consistent with the turnover rate of gangliosides, which in rat brain is reported to be highest between 10 and 20 days.
Effects of cocaine on the rat cerebral commissure
Authors: Ojima Kazuya , Abiru Hitoshi , Fukui Yuko
Published: July 2002

We investigated the effects of cocaine on the corpus callosum, the nerve fibre bundle that connects the bilateral cerebral hemispheres. Our experiments in rats confirmed that, in the control group, the mid‐sagittal area of the corpus callosum in the adult male was significantly larger than this area in the female. Early postnatal exposure to cocaine abolished this sexual dimorphism, that is, cocaine‐treated males had a significantly smaller callosal area than the control males. Cocaine induced no significant changes in the weight of the body or brain. There were no significant sex differences in the midline sagittal area of the anterior commissure, and no apparent effects of cocaine exposure were determined in this structure. These findings suggest that early postnatal exposure to cocaine abolishes the sexual differentiation of the corpus callosum in male rats.
Emotional family atmosphere and relapse: investigations on the role of relapse definition, duration of illness and resignation of relatives
Authors: Schulze Mönking H , Buchkremer G
Published: July 2002

An analysis of existing studies on the expressed emotion (EE) concept shows that the predictive validity of the EE index depends on the definition of relapse. Whilst symptomatic relapses are recorded in patients from high-EE families, about 3 times as often as in patients from low-EE families, a comparison of hospitalization rates reveals a difference of only approximately 1.5 times. In the Münster Families Study, a prospective intervention study to evaluate the therapeutic impact of relatives' groups, the predictive value of the familial emotional atmosphere could be measured in a prospective design. Ninety-nine DSMIII Schizophrenics, 72 male, 27 female, mean age 27 (SD 6.6), mean duration of illness 5.5 (SD 4.3) were followed up. In the first eighteen months patients from high-EE- families had a risk of rehospitalisation approximately 1.5 (p < 0.05). Further analysis showed that relapse rates among patients from high-EE and low-EE families were roughly the same for patients with a duration of illness of less than 4.5 years but were significantly different after a longer duration (p = 0.019). Besides a high EE rating, resignation of relatives was also a significant (p = 0.000) predictor of rehospitalisation for high and — even more — low EE families. Possible causes of these findings and therapeutic consequences are discussed.
In vivo localisation and stability of human Mcl‐1 using green fluorescent protein (GFP) fusion proteins
Authors: Akgul Cahit , Moulding Dale A , White M.R.H , Edwards Steven W
Published: July 2002

Mcl‐1 is an anti‐apoptotic member of the Bcl‐2 family of proteins. We have expressed full length and mutated GFP:Mcl‐1 fusion proteins to define structural motifs that control protein localisation and stability. When expressed in U‐937 cells, full length Mcl‐1 locates primarily within mitochondria and its half‐life was approximately 3 h, which was identical to the native, endogenously expressed protein. When the terminal 20 amino acids from the C‐terminus of the protein were detected, the protein was diffused in the cytoplasm, but its stability was unaffected. This confirms that this region is responsible for efficient targeting to mitochondria. Surprisingly, deletion of 104 amino acids (residues 79–183) that contain putative PEST sequences and other stability regulating motifs, did not affect protein stability.
A chloroplastic RNA‐binding protein is a new member of the PPR family
Authors: Lahmy Sylvie , Barnèche Fredy , Derancourt Jean , Filipowicz Witold , Delseny Michel , Echeverria Manuel
Published: July 2002

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA‐binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.
Oligomerization of pro‐opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway
Authors: Cawley Niamh X , Normant Emmanuel , Chen Anthony , Loh Y.Peng
Published: July 2002

Studies indicate that pro‐opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane‐bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73–83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self‐associate to form oligomers, prior or subsequent to binding to CPE. Using cross‐linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. ΔN‐POMC, which lacks the N‐terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.
Direct binding of hepatitis B virus X protein and retinoid X receptor contributes to phosphoenolpyruvate carboxykinase gene transactivation
Authors: Kong Hee Jeong , Hong Sun Hwa , Lee Min Young , Kim Han Do , Lee Jae Woon , Cheong JaeHun
Published: July 2002

The X gene product of the human hepatitis B virus (HBx), a major factor responsible for hepatitis and hepatocellular carcinoma, modulates transactivation by a variety of transcription factors. Herein, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was found to be regulated transcriptionally by HBx through two distinct promoter regions. The cAMP response element (CRE)‐1 site within the proximal promoter region mediated the HBx‐induced transactivation of the PEPCK gene through C/EBPα and ATF‐2. A retinoid X receptor (RXR) response element within the distal promoter region also contributed to the HBx‐induced transactivation. Consistent with these results, HBx directly interacted with RXR, and the interaction interfaces were localized to the transactivation domain of HBx and the ligand binding domain of RXR.
Domain architecture of a Caenorhabditis elegans AKAP suggests a novel AKAP function
Authors: Herrgård Sanna , Jambeck Per , Taylor Susan S. , Subramaniam Shankar
Published: July 2002

A‐kinase anchoring proteins (AKAPs) are adapter proteins that are involved in directing cAMP‐dependent protein kinase and some other signaling enzymes to certain intracellular locations. In this study, we investigate the domain architecture of an AKAP from Caenorhabditis elegans (AKAPCE). We show that AKAPCE shares two domains with the Smad anchor for receptor activation, a FYVE‐finger and a transforming growth factor β (TGFβ) receptor binding domain, suggesting that AKAPCE may interact with a receptor belonging to the TGFβ receptor family. This predicted novel AKAP function supports the recent view of AKAPs as adapter proteins that can be involved in various signaling pathways.
5S rRNA binding proteins from the hyperthermophilic archaeon, Pyrococcus furiosus
Authors: Furumoto Hiromi , Taguchi Atsuo , Itoh Takashi , Morinaga Tsutomu , Itoh Takuzi
Published: July 2002

A determination was made of the nucleotide sequence of the 2719 bp region of a ribosomal protein gene cluster (PfeL32–PfeL19–PfL18–PfS5–PfL30) containing a 5S rRNA binding protein L18 homolog of hyperthermophilic archaea Pyrococcus furiosus. The organization of the archaeal ribosomal protein gene cluster is similar to that in the spc‐operon of Escherichia coli (L6‐L18‐S5‐L30‐L15) but has two additional genes, namely those encoding PfeL32 and PfeL19, which were identified as extra proteins that are apparently not present in bacterial E. coli. Using an inducible expression system, P. furiosus mature PfL18 protein and a mutant PfL18 with the basic N‐terminal amino acid region deleted were produced in large amounts in E. coli and Northwestern analysis showed the N‐terminal region of PfL18, including the conserved arginine‐rich region, to have a significant role in 5S rRNA–PfL18 interaction.
Molecular control of neutrophil apoptosis
Authors: Akgul Cahit , Moulding Dale A , Edwards Steven W
Published: July 2002

Human neutrophils constitutively undergo apoptosis and this process is critical for the resolution of inflammation. Whilst neutrophil apoptosis can be modulated by a wide variety of agents including GM‐CSF, LPS and TNF‐α, the molecular mechanisms underlying neutrophil death and survival remain largely undefined. Recent studies have shown the involvement of members of the Bcl‐2 protein family (especially Mcl‐1 and A1) and caspases in the regulation and execution of neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen‐activated protein kinases, also play critical roles in transducing the signals that result in neutrophil apoptosis or extended survival. This review summarises current knowledge on the molecular mechanisms and components of neutrophil apoptosis.
A synaptojanin‐homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation
Authors: Marcus Sandra L. , Wenk Markus R. , Steele-Mortimer Olivia , Finlay B.Brett
Published: July 2002

The Ser–Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5‐biphosphate and phosphatidylinositol 3,4,5‐triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3‐phosphate was not a substrate, suggesting the 5′ phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5‐phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a ΔsigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl‐terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.
Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli
Authors: Vishnuvardhan Daesety , Kakiuchi Nobuko , Urvil Petri T , Shimotohno Kunitada , Kumar P.K.R , Nishikawa Satoshi
Published: July 2002

The serine protease domain of HCV comprising amino acids 1027–1218 (ΔNS3) was expressed in E. coli with a His tag at its N‐terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (∼3 mg/l of cultured cells). The ΔNS3 efficiently cleaves a 17‐mer peptide corresponding to the NS5A‐NS5B junction with k
cat/K
m=160×10−3 min−1 μM−1 in the presence of NS4A peptide. Our ΔNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The ΔNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203–2506 amino acids) that was synthesized by in vitro transcription and translation system.
Terminal oxidases of Azoarcus sp. BH72, a strictly respiratory diazotroph
Authors: Reinhold-Hurek Barbara , Zhulin Igor B.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Amino acid substitutions in the first transmembrane domain (TM1) of P‐glycoprotein that alter substrate specificity
Authors: Taguchi Yoshitomo , Morishima Masaki , Komano Tohru , Ueda Kazumitsu
Published: July 2002

Recently, we showed that the amino acid at position 61 in TM1 of human P‐glycoprotein is important in deciding the substrate specificity of this protein. In this work, we investigated whether the amino acids other than His61 in TM1 of P‐glycoprotein are also essential in the function of this protein. Nine amino acids residues, from Ala57 to Leu65 in TM1, were independently substituted to Arg, and analyzed the drug resistance of cells stably expressing each of these mutant P‐glycoproteins. The mutant P‐glycoproteins Ile60→Arg, His61→Arg, Ala63→Arg, Gly64→Arg, and Leu65→Arg were normally processed and expressed in the plasma membrane. Substrate specificities of mutant P‐glycoproteins Gly64→Arg and Leu65→Arg were quite different from that of the wild type, and similar to that of the His61→Arg mutant, while the Ile60→Arg and Ala63→Arg mutant P‐glycoproteins showed similar substrate specificities to that of the wild‐type P‐glycoprotein, suggesting that not only the amino acid residue at position 61 but also those at position 64 and 65 are also important in deciding the substrate specificity of P‐glycoprotein. These three amino acids His61, Gly64, and Leu65 would form a compact region on an α‐helix arrangement of TM1. These results suggest that a region consisting of His61, Gly64, and Leu65 in TM1 would participate in the formation of the recognition site for substrates of P‐glycoprotein.
Biogenesis of COPI‐coated transport vesicles
Authors: Nickel Walter , Wieland Felix T
Published: July 2002

Biosynthetic protein transport and sorting along the secretory pathway represents the last step in biosynthesis of a variety of proteins. Proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER towards their final destinations. The successive compartments of the secretory pathway are connected by vesicular shuttles that mediate delivery of cargo. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has also been identified. This review is focused on COPI‐coated vesicles that operate in protein transport within the early secretory pathway. Rather than representing a general overview of the role of COPI‐coated vesicles, this mini‐review will discuss mechanisms specifically related to the biogenesis of COPI‐coated vesicles: (i) a possible role of phospholipase D in the formation of COPI‐coated vesicles, (ii) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly, and (iii) the direction COPI‐coated vesicles may take within the early secretory pathway.
Two‐dimensional distribution of Gi2α in the plasma membrane: a critical evaluation by immunocytochemistry
Authors: Nomura Ryuji , Inuo Chisato , Takahashi Yukiko , Asano Tomiko , Fujimoto Toyoshi
Published: July 2002

Caveolae have been postulated as a center for signal transduction, because many signaling molecules are concentrated in caveolin‐rich fractions. We took Gi2α as an example and examined whether it is constitutively concentrated in caveolae. First, the behavior of caveolin and Gi2α in density‐equilibrium ultracentrifugation was reexamined. By collecting fractions efficiently, caveolin and Gi2α were found to distribute differently. Secondly, by novel immunocytochemical methods it was found that the labeling density of Gi2α was 2.29 times higher in caveolae than in the non‐caveolar plasma membrane. The results indicate that the concentration of Gi2α in caveolae is lower than deduced from most biochemical studies.
Intracellular Ca2+ dependence of nitric oxide mediated enhancement of interleukin‐8 secretion in human endothelial cells
Authors: Volk Thomas , Hensel Mario , Mäding Karsten , Egerer Karl , Kox Wolfgang J.
Published: July 2002

Nitric oxide (NO⋅) can induce transient [Ca2+] changes in endothelial cells not different from receptor mediated signalling. Whether this Ca2+ signal may provide a link with IL‐8 secretion induced by NO⋅ donors was investigated in human endothelial cells. Sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl‐dl‐penicillamine (SNAP) dose dependently increased IL‐8 production in this cell type. Additive IL‐8 secretion was found with TNFα. Buffering intracellular Ca2+ with MAPT/AM suppressed NO⋅ induced [Ca2+]i changes and reduced subsequent IL‐8 secretion. The additive effect of both NO⋅ donors on TNFα induced IL‐8 secretion was completely blocked in the presence of MAPT/AM. SKF 96365, which has been shown to block receptor mediated Ca2+ entry, and TMB‐8, which blocks intracellular Ca2+ release, both inhibited IL‐8 secretion, particularly when TNFα was used as a costimulator, indicating that [Ca2+]i changes are important components of IL‐8 induction by NO⋅.
Insertion of a SNS‐specific tetrapeptide in S3–S4 linker of D4 accelerates recovery from inactivation of skeletal muscle voltage‐gated Na channel μ1 in HEK293 cells
Authors: Dib-Hajj S.D , Ishikawa K , Cummins T.R , Waxman S.G
Published: July 2002

Na channel subunits αSNS (PN3) and αμ1(SkM1) produce slowly inactivating/TTX‐resistant and rapidly inactivating/TTX‐sensitive currents, respectively. αSNS (PN3) current recovers from inactivation (reprimes) rapidly. Sequence alignment identified the tetrapeptide SLEN, in the S3–S4 linker of D4, as αSNS‐specific. To determine whether SLEN endows Na channels with slow kinetics and/or rapid repriming, we analyzed the transient Na current produced by a chimera μ1SLEN in HEK293 cells. Neither kinetics nor voltage dependence of activation and inactivation was affected. However, repriming was twice as fast as in the wild type at −100 mV. This suggests that SLEN may contribute to the rapid repriming of TTX‐resistant Na current.
Chain termination codons and polymerase‐induced frameshift mutations
Authors: Jestin Jean-Luc , Kempf Achim
Published: July 2002

The consensus sequence for single‐base deletions in non‐reiterated runs during in vitro DNA‐dependent DNA polymerisation is refined using data available in the literature. This leads to the observation that chain termination codons are hotspots for single‐base deletions. The evolutionary implications are discussed in two models which differ in whether polymerases evolved while the genetic code emerged or after the genetic code was fixed. A possible answer to the question `Why are stop codons just what they are?' is suggested.
Sequence and characterisation of the RET proto‐oncogene 5′ flanking region: analysis of retinoic acid responsiveness at the transcriptional level
Authors: Patrone Giovanna , Puliti Aldamaria , Bocciardi Renata , Ravazzolo Roberto , Romeo Giovanni
Published: July 2002

The RET proto‐oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up‐regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5′ to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.
Involvement of calmodulin in the activation of store‐operated Ca2+ entry in rat hepatocytes
Authors: Cao Yumei , Chatton Jean-Yves
Published: July 2002

The possible participation of calmodulin in the activation of store‐operated Ca2+ entry (SOC) in single rat hepatocytes was investigated microspectrofluorimetrically. SOC was triggered after discharging intracellular Ca2+ stores using the endoplasmic reticulum Ca2+‐ATPase inhibitor thapsigargin in the absence of external Ca2+. Re‐admission of bath Ca2+ caused a rapid and pronounced Ca2+ entry. The calmodulin antagonists calmidazolium or CGS 9343B applied before the thapsigargin treatment inhibited SOC, whereas they were ineffective when added after the thapsigargin‐induced Ca2+ transient. This study suggests that activation of calmodulin after the elevation of cytosolic Ca2+ associated with the emptying of Ca2+ stores is involved in the triggering of SOC in hepatocytes.
Non‐glycosylated human B7‐1(CD80) retains the capacity to bind its counter‐receptors1
Authors: Chen Aoshuang , Meyerson Howard J. , Salvekar Anupama , Tykocinski Mark L.
Published: July 2002

Though the cell surface‐associated costimulator B7‐1(CD80) is known to be highly N‐glycosylated, the functional significance of this N‐glycosylation has not been evaluated. Two experimental approaches were taken to assess the influence of N‐glycosylation on human B7‐1 function. First, stable K562 transfectants expressing human B7‐1 were treated with the N‐glycosylation inhibitor tunicamycin. This treatment reduced the levels of B7‐1 at the cell surface as judged by both indirect immunofluorescence/flow cytometry and immunoprecipitation analyses. Significantly, the non‐glycosylated cell surface‐associated B7‐1 on tunicamycin‐treated cells retained the capacity to bind CTLA‐4·Ig, a soluble derivative of the CTLA‐4(CD152) counter‐receptor. Second, experiments were performed with bacterially‐produced non‐glycosylated derivatives of human B7‐1, comprising either the complete B7‐1 extracellular domain (hB7‐1·ed) or the membrane‐proximal IgC‐homologue domain of B7‐1 in isolation (hB7‐1·IgC). While the hB7‐1·IgC derivative failed to bind to CTLA‐4, the larger hB7‐1·ed derivative associated with CTLA‐4·Ig in cell‐free binding assays. Futhermore, recombinant hB7‐1·ed effectively blocked B7‐1‐mediated costimulation in an in vitro T cell proliferation assay, suggesting that this soluble non‐glycosylated B7‐1 derivative is capable of engaging CD28, the B7 counter‐receptor implicated in T cell activation. Taken together, these data indicate that the N‐glycosylation of B7‐1 is not required for its association with counter‐receptors. Moreover, the findings pave the way for the therapeutic use of recombinant bacterial B7‐1 derivatives as competitive inhibitors of B7‐mediated signals.
Ectocellular CD38‐catalyzed synthesis and intracellular Ca2+‐signalling activity of cyclic ADP‐ribose in T‐lymphocytes are not functionally related
Authors: da Silva Cristina P , Schweitzer Katrin , Heyer Petra , Malavasi Fabio , Mayr Georg W , Guse Andreas H
Published: July 2002

Cyclic ADP‐ribose (cADPR) is a natural metabolite of β‐NAD+ with a potent Ca2+‐mobilizing activity in different cell types, including T‐lymphocytes. We investigated (i) whether stimulation of T‐lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP‐ribosyl cyclase and cADPR‐hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca2+‐mobilizing effects of this nucleotide in Jurkat and HPB.ALL T‐lymphocytes. The anti‐CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca2+ concentration ([Ca2+]i). In contrast, activation of an ectocellular ADP‐ribosyl cyclase by preincubation of cells with β‐NAD+ led to a dose‐dependent increase in cADPR, but no changes in [Ca2+]i were observed. However, extensive washing of the cells following preincubation with NAD+ demonstrated that the increases in cADPR were not intracellular but due to cell surface‐associated nucleotide. Accordingly, measurements of ADP‐ribosyl cyclase activity in intact T‐cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP‐ribosyl cyclase activity of CD38 on the regulation of the cADPR‐mediated intracellular Ca2+‐signalling in T‐lymphocytes.
Molecular analysis of a new splice variant of the human melanocortin‐1 receptor
Authors: Tan Carina P , Kulju McKee Karen , Weinberg David H , MacNeil Tanya , Palyha Oksana C , Feighner Scott D , Hreniuk Donna L , Van Der Ploeg Lex H.T , MacNeil Douglas J , Howard Andrew D
Published: July 2002

The primary hormonal regulator of pigmentation is melanocyte stimulating hormone derived from proopiomelanocortin by proteolytic processing. The melanocortin‐1 receptor serves a key role in the regulation of pigmentation. We describe the identification of the first intron within a melanocortin receptor. A new melanocortin‐1 receptor isoform, generated by alternative mRNA splicing, encodes an additional 65 amino acids at the predicted intracellular, C‐terminal tail of the melanocortin‐1 receptor. When expressed in heterologous cells, the new spliced form of the melanocortin‐1 receptor (melanocortin‐1 receptor B) appears pharmacologically similar to the non‐spliced melanocortin‐1 receptor. Melanocortin‐1 receptor B is expressed in testis, fetal heart and melanomas.
Two distinct translocation intermediates can be distinguished during protein transport by the TAT (Δph) pathway across the thylakoid membrane
Authors: Berghöfer Jürgen , Klösgen Ralf Bernd
Published: July 2002

During thylakoid transport of the chimeric precursor protein 16/23 which takes place by the twin arginine translocation (TAT) (Δph)‐dependent pathway, two distinct translocation intermediates can be identified which represent successive steps in the translocation process. Both intermediates are partially inserted into the thylakoid membrane and can be distinguished by specific degradation fragments occurring after thermolysin treatment of the thylakoids. While the formation of the early translocation intermediate does not depend on a functional TAT translocation machinery, the appearance of the late intermediate is strictly coupled to the Δph‐dependent transport of the 16/23 chimera. Accordingly, this translocation intermediate is found associated with two distinct complexes in the thylakoid membrane having apparent molecular masses of approximately 560 and 620 kDa, respectively.
Effect of sphingosylphosphorylcholine on the single channel gating properties of the cardiac ryanodine receptor
Authors: Uehara Akira , Yasukochi Midori , Imanaga Issei , Berlin Joshua R.
Published: July 2002

The effects of the lysosphingolipid, sphingosylphosphorylcholine (SPC), on the cardiac ryanodine receptor (RyR) were examined. The open probability of cardiac RyR incorporated in lipid bilayers was decreased by cytoplasmic, but not lumenal side application of micromolar concentrations of SPC. Modification of channel function was characterized by the appearance of a long‐lived closed state in addition to the brief channel closings observed in the presence and absence of SPC. Open channel kinetics and ion conduction properties, however, were not altered by this compound. These results suggest that SPC, a putative second messenger derived from sphingomyelin, may regulate Ca2+ release from the sarcoplasmic reticulum by modifying the gating kinetics of the RyR.
Erythropoietin, granulocyte‐colony stimulating factor, interleukin‐1β and interleukin‐6 during the normal menstrual cycle
Authors: Makinoda S , Mikuni M , Sogame M , Kobamatsu Y , Furuta I , Yamada H , Yamamoto R , Fujimoto S
Published: July 2002

Objective: Menstruation and ovulation are the main events during the menstrual cycle. Menstruation may influence the complete blood count because of its blood loss. Since ovulation is an inflammatory‐like phenomenon, white blood cell count may change during ovulation. For these reasons, we have investigated the complete blood count and peripheral serum concentrations of associated cytokines during the normal menstrual cycle. Materials and methods: Normal healthy female volunteers (N = 9; age, 21–33 years) with menstrual cycles of 25–33 days recorded basal body temperature every day and venous blood samples were collected three times per week. Complete blood count was performed and erythropoietin, granulocyte‐colony stimulating factor, interleukin‐1β, interleukin‐6 and hormones (FSH, LH, estradiol and progesterone) were measured. Results: The red blood cell, total white blood cell, granulocyte and platelet counts showed no statistically significant changes during the menstrual cycle when using analysis of variance with the Scheffe F‐test. No changes in erythropoietin, interleukin‐1β and interleukin‐6 were seen throughout the menstrual cycle. In contrast, serum granulocyte‐colony stimulating factor concentrations increased significantly to 28.0 ± 3.1 pg/ml (mean ± S.E.M.) during the ovulatory phase, compared to 10.7 ± 0.6 pg/ml in other phases. Conclusion: These results indicate that menstrual blood loss does not affect the complete blood count and suggest that granulocyte‐colony stimulating factor plays an important role in the mechanism of ovulation.
Contraceptive methods: potential for abuse
Authors: Benagiano G. , Cottingham J.
Published: July 2002

Contraceptive technologies are one of the most extraordinary advances of the twentieth century, enabling women and men to prevent unwanted pregnancy. They have also been criticized for giving rise to abusive practices in the name of population control. Some people have even argued that contraceptive methods which have a potential for abuse should not be developed. We maintain that eliminating research on such methods does not address the problem of abuse. Abuse has been defined as the violation of the right to liberty and security of the person, which requires that an individual's free and informed consent be given before research is done on, or treatment is provided to, that individual. A number of international guidelines on the ethical conduct of research involving human subjects, and on the provision of medical services, emphasise the importance of informed consent as part of informed decision‐making of individuals. Irrespective of the contraceptive methods already available or under development, much public education is needed to ensure that investigators, health providers, users of services and research participants fully understand what informed consent means, what it implies and how it should protect people. Non‐governmental organizations and others can use the various national and international mechanisms that exist to investigate cases of alleged abuse and to bring redress in such situations. The international community has a duty to foster and strengthen ethical practices in research and service provision of contraceptive methods.
Transcutaneous electrical nerve stimulation (TENS) for adjuvant pain‐relief during labor and delivery
Authors: Kaplan B , Rabinerson D , Lurie S , Bar J , Krieser U.R , Neri A
Published: July 2002

Objective: We examined the efficacy of transcutaneous electrical nerve stimulation (TENS) in general and the new Freemom TENS device (LifeCare, Israel) in particular, for pain relief during labor and delivery. Methods: The study group consisted of 104 women. Forty‐six nulliparas (44.2%) and 58 multiparas (55.8%), all of whom used the TENS device for pain relief during labor. All participants completed a questionnaire on the degree of pain relief afforded them by TENS during the delivery and related questions. The objective evaluation was based on the documented labor and delivery parameters including medical interventions during delivery. Results: The majority of subjects (72% of the nulliparas and 69% of the multiparas) considered TENS effective for the relief of pain during labor. Most of them (67% of the nulliparas and 60% of the multiparas) responded positively to the use of TENS in future deliveries. Sixty‐five percent of the multiparas considered TENS at least as effective as the other pain relief methods they had used before. TENS significantly reduced the duration of the first stage of labor P<0.001 for nulliparas, P<0.005 for multiparas and it significantly decreased the amount of analgesics administered to individual patients. No significant difference was found in fetal heart rate tracings, Apgar scores and cord blood pH between the study group and an equal number of matched controls who used other forms of pain management. Conclusions: TENS is an effective non‐pharmacological, non‐invasive adjuvant pain relief modality for use in labor and delivery. TENS application reduced the duration of the first stage of labor and the amount of analgesic drug administered. There were no adverse effects on mothers or newborns.
Obstetric outcome of immigrants from mainland China in Hong Kong
Authors: Leung T.N. , Lau T.K. , Roach V.J. , Wilson D. , Rogers M.S. , Chang A.M.Z.
Published: July 2002

Objective: To compare obstetric outcomes in two socio‐economically distinct populations; illegal immigrants, who travelled across the Chinese border near term to deliver in Hong Kong between 1994 and 1996, and local Hong Kong residents. Methods: We identified two obstetric populations in our hospital with distinct social characteristics and patterns of antenatal care. A retrospective study of pregnancy outcome was conducted in these women. Results: When compared with Hong Kong residents, the illegal immigrants had a significantly lower incidence of antenatal complications, a lower requirement for obstetric intervention, and comparable obstetric outcome with the exception of a higher rate of delivery before arrival at hospital. Conclusion: Our findings were in contrast to previous reports, and contrary to our expectations. Obstetric outcome is multifactorial and previous assumptions about unbooked or socially disadvantaged patients should be re‐evaluated.