bluuebunny/crossref_metadata_2025 huggingface dataset:
Raft and cytoskeleton associations of an ABC transporter: P‐glycoprotein
Authors: Bacso Zsolt , Nagy Henrietta , Goda Katalin , Bene László , Fenyvesi Ferenc , Matkó János , Szabó Gábor
Published: August 2004

Background

A novel flow cytometric assay has been described in an accompanying report (Gombos et al.,

Methods

The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft‐preserving Triton X‐100 or Nonidet P‐40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft‐mediated and non–raft‐related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents.

Results

The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2‐labeled cell‐surface Pgps to Triton X‐100 versus Nonidet P‐40. Approximately 34% of the UIC2 Fab‐labeled Pgp molecules were associated with the cytoskeleton through detergent‐resistant, cholesterol‐sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and GM1‐gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping.

Conclusions

By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS‐174‐T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels. © 2004 Wiley‐Liss, Inc.
Analysis of translocations that involve the NUP98 gene in patients with 11p15 chromosomal rearrangements
Authors: Kobzev Yuri N. , Martinez‐Climent Jose , Lee Sanggyu , Chen Jianjun , Rowley Janet D.
Published: August 2004

The NUP98 gene has been reported to be fused with at least 15 partner genes in leukemias with 11p15 translocations. We report the results of screening of cases with cytogenetically documented rearrangements of 11p15 and the subsequent identification of involvement of NUP98 and its partner genes. We identified 49 samples from 46 hematology patients with 11p15 (including a few with 11p14) abnormalities, and using fluorescence in situ hybridization (FISH), we found that NUP98 was disrupted in 7 cases. With the use of gene‐specific FISH probes, in 6 cases, we identified the partner genes, which were PRRX1 (PMX1; in 2 cases), HOXD13, RAP1GDS1, HOXC13, and TOP1. In the 3 cases for which RNA was available, RT‐PCR was performed, which confirmed the FISH results and identified the location of the breakpoints in patient cDNA. Our data confirm the previous findings that NUP98 is a recurrent target in various types of leukemia. © 2004 Wiley‐Liss, Inc.
Benzo[a]pyrene diol epoxide‐induced 9p21 aberrations associated with genetic predisposition to bladder cancer
Authors: Hazra Aditi , Grossman H. Barton , Zhu Yong , Luo Sherry , Spitz Margaret R. , Wu Xifeng
Published: August 2004

CDKN2A, 9p21, encodes two alternatively spiked, functionally distinct, tumor‐suppressor proteins, P16INK4A and P14ARF, which play active roles in the RB1 and TP53 pathways, respectively. Deletion of 9p is one of the most frequent genomic alterations in bladder cancer. In addition, alterations of 9p21 and P16 are frequently seen in the epithelial cells of chronic smokers. This pilot study evaluated whether 9p21 aberrations induced by exposure in vitro to benzo[a]pyrene diol epoxide (BPDE), the metabolic product of benzo[a]pyrene, a constituent of tobacco smoke, were more common in the peripheral blood lymphocytes of 61 bladder cancer patients compared to 64 matched controls. Our hypothesis was that 9p21 sensitivity to BPDE reflects the susceptibility of a specific locus to damage from carcinogens in tobacco smoke. We found that BPDE‐induced chromosome band 9p21 aberrations were significantly higher in lymphocytes of bladder cancer cases (24.97 ± 5.26 per 1,000) than in controls (20.72 ± 4.51 per 1,000; P < 0.0001). However, no difference was observed for CEP9, a control locus. After adjustment for age, sex, ethnicity, and smoking status, 9p BPDE sensitivity had an odds ratio (OR) of 9.01 [95% confidence interval (95% CI) 3.75, 21.67] for bladder cancer. We further observed a gradient of elevated bladder cancer risk associated with increasing chromosomal damage. The adjusted ORs for subjects in the second, third, and highest quartiles of BPDE‐induced 9p21 aberrations relative to the first quartile were 0.48 (0.04, 5.69), 5.14 (1.12, 23.59), and 21.51 (4.75, 97.34), respectively, providing increasing dose–response evidence of the locus‐specific alterations. Thus, 9p21 may be a molecular target for BPDE‐induced damage in bladder cancer cases. © 2004 Wiley‐Liss, Inc.
Specific chromosome aberrations in peripheral blood lymphocytes are associated with risk of bladder cancer
Authors: Shao Lina , Lerner Seth L. , Bondaruk Jolanta , Czerniak Bogdan A. , Zeng Xiaomin , Grossman H. Barton , Spitz Margaret R. , Wu Xifeng
Published: August 2004

Specific chromosome aberrations in peripheral blood lymphocytes (PBLs) in chromosomes 9 and 11 may be associated with bladder cancer. To investigate this hypothesis, in this study, we used a whole‐chromosome painting technique to detect chromosomal aberrations in PBLs from 100 patients with bladder cancer and 100 matched controls. We also used a locus‐specific fluorescence in situ hybridization technique to study 9p21 and cyclin D1 gene (CCND1) aberrations in PBLs of 10 patients and 10 controls and in tumor tissues of 38 additional cases. The chromosome‐painting analysis showed that there were more aberrations of chromosomes 9 and 11 in bladder cancer patients than in controls. When categorized by type, the number of deletions of 9p and of translocations of chromosome 11 was significantly higher in patients than in controls (P < 0.05). Stratified analysis showed a larger odds ratio (OR) for bladder cancer in individuals with either a 9p deletion or a chromosome 11 translocation/amplification and an even larger OR in individuals with both aberrations. Using locus‐specific analysis, we found that 9p21 aberrations occurred more frequently in bladder cancer patients (12.1 per 1,000 interphase cells) than in controls (6.4 per 1,000 interphase cells, P < 0.05); CCND1 translocation and amplification were observed only in bladder cancer patients. Tumor tissue analysis showed that aberrations of 9p21 (40.0 per 100 interphase cells) and CCND1 (43.8 per 100 interphase cells) were very common. Thus, we concluded that 9p deletions and translocations of chromosome 11, especially at 9p21 and CCND1, are associated with bladder cancer. © 2004 Wiley‐Liss, Inc.
Stereoelectronic Effects in the Iodine‐Promoted Oxidation of Pentacyclic Tetrahydroisoquinolines
Authors: Koepler Oliver , Laschat Sabine , Miehlich Burkhard , Baro Angelika , Fischer Peter
Published: August 2004

Iodine‐promoted oxidations of the pentacyclic tetrahydroisoquinolines 4a, 4b, and 10 were investigated. Whereas the all‐cis diastereoisomer 4a containing an arylamino moiety gave the iminium ion 5 as the primary product, which subsequently underwent intramolecular aminal formation to 6, the corresponding all‐trans diastereoisomer 4b epimerized to the all‐cis diastereoisomer 4a via iminium ion 7. In contrast, tetrahydroisoquinoline 10 could be cleanly oxidized to the corresponding isoquinolizidinium ion 11. Mechanistic considerations were supported by molecular‐modeling calculations.
An Effective and Convenient Method for the Preparation of KAD‐1229
Authors: Liu Jianchao , Yang Yushe , Ji Ruyun
Published: August 2004

A new convenient method for the asymmetric synthesis of the potent hypoglycemic agent KAD‐1229 was developed. The key step of this method is diasteroselective alkylation of 1 to give crude 2 (d.e.>93%) in good yield with the easily available Oppolzer's camphorsultam as chiral auxiliary. The overall yield of the product was 57%.
Synthesis, Spectroscopic Studies, and Crystal Structures of Phenylorganotin Derivatives with [Bis(2,6‐dimethylphenyl)amino]benzoic Acid: Novel Antituberculosis Agents
Authors: Dokorou Vaso , Kovala‐Demertzi Dimitra , Jasinski Jerry P. , Galani Angeliki , Demertzis Mavroudis A.
Published: August 2004

The novel triphenyl adduct of 2‐[(2,6‐dimethylphenyl)amino]benzoic acid (HDMPA; 1), i.e., [SnPh3(DMPA)] (2), the dimeric tetraorganostannoxane [Ph2(DMPA)SnOSn(DMPA)Ph2]2 (3), and the monomeric adduct [SnPh2(DMPA)2] (4), where DMPA is monodeprotonated HDMPA, have been prepared and structurally characterized by means of IR, 1H‐NMR, and 13C‐NMR spectroscopy. The structures of 1 and 2 have been determined by X‐ray crystallography. Single‐crystal X‐ray‐diffraction analysis of 1 revealed that there are two molecules in the asymmetric unit, HD1 and HD2, differing in conformation, both forming centrosymmetric dimers linked by H‐bonds between the carboxylic O‐atoms. X‐Ray analysis of 2 revealed a pentacoordinate structure containing Ph3Sn coordinated to the carboxylato group. Significant CH/π interactions and intramolecular H‐bonds stabilize the structures of 1 and 2, which self‐assembled via CH/π and π/π‐stacking interactions. The Ph3Sn adduct 2 was found to be a promising antimycobacterial lead compound, displaying activity against Mycobacterium tuberculosis H37Rv. The cytotoxiciy in the Vero cell line is also reported.
Two Novel ent‐Abietane Diterpenoids from Isodon xerophilus
Authors: Niu Xue‐Mei , Li Sheng‐Hong , Na Zhi , Lin Zhong‐Wen , Sun Han‐Dong
Published: August 2004

The tow novel ent‐abietane diterpenoids 1 and 2, named xerophilusins R and S, respectively (=(11β)‐6,11‐dihydroxy‐7,20‐dioxo‐ent‐abieta‐5,8(14),15(17)‐trien‐16‐oic acid δ‐lactone and (11β,15S)‐6,11,17‐trihydroxy‐7,20‐dioxo‐ent‐abieta‐5,8(14)‐dien‐16‐oic acid δ‐lactone, resp.), were isolated from Isodon xerophilus (C. J. Wu et H. W. LI) H. Hara. Their structures were established by spectroscopic means.
Lycopodine‐Type Lycopodium Alkaloids from Huperzia serrata
Authors: Tan Chang‐Heng , Zhu Da‐Yuan
Published: August 2004

Eleven Lycopodium alkaloids with a lycopodine‐type skeleton were isolated from the basic material of the whole plant of Huperzia serrata (Thunb.) Trev. (Huperziaceae). Among them, 12‐epilycodoline N‐oxide (=(12α,15R)‐12‐hydroxy‐15‐methyllycopodan‐5‐one N‐oxide; 1), 7‐hydroxylycopodine (=(15S)‐7‐hydroxy‐15‐methyllycopodan‐5‐one; 2), and 4,6α‐dihydroxylycopodine (=(6α,15R)‐4,6‐dihydroxy‐15‐methyllycopodan‐5‐one; 3) are new compounds. Their structures were identified spectroscopically, especially by means of 1D‐ and 2D‐NMR.
Thermal Isomerization of Isoborneols and Dehydroisoborneols to New Chiral Building Blocks in Terpenoid Synthesis
Authors: Rüedi Georg , Hansen Hans‐Jürgen
Published: August 2004

The substituted isoborneols 1a–1g and 5,6‐dehydroisoborneols 6a–6c, readily prepared in excellent yields from (+)‐camphor and (+)‐5,6‐dehydrocamphor (2) by aryl, vinyl, or alkyl Grignard addition in the presence of stoichiometric amounts of CeCl3, were thermally isomerized in a flow reactor system under DGPTI (dynamic gas‐phase thermo‐isomerization) conditions at temperatures between 480 and 630° to give the enantiomerically pure monocyclic carbonyl compounds 7a–7d, 19a, b, 23, and 24. In all cases, product formation proceeded highly regio‐ as well as stereoselectively. The absolute configurations of the new stereogenic centers were determined by 1H‐NOE measurements. DGPTI of the aryl substrates 1a–1d is proposed to effect initial cleavage of the weakest single bond in the molecule under formation of a diradical intermediate state followed by intramolecular H‐abstraction to afford the acetophenone derivatives 7a–7d. This reaction path was further supported by a 2H‐labeling study showing the OH group to be the exclusive H‐source. In contrast, DGPTI of the vinyl substrates 1e and 6b allowed concerted retro‐ene and oxy‐Cope rearrangements. In the case of 5,6‐dehydro‐2‐phenylisoborneol (6a), concomitant diradical and retro‐Diels–Alder reaction pathways could be observed. In addition, a new route to (+)‐trans‐α‐campholanic acid (9) and (+)‐trans‐α‐dihydrocampholytic acid (14) is presented by regioselective Baeyer–Villiger oxidation and subsequent hydrolysis of 7c and 7d, respectively.
Ozonolyse von Enolethern. 10. Mitteilung
Authors: Schank Kurt , Beck Horst , Pistorius Susanne
Published: August 2004

Ozonolysis of Enol Ethers. Part 10. Ozonization of Enol Ethers from 1,2‐ and 1,3‐Dicarbonyl Compounds: Direct Quantitative Synthesis of Phthalonic Acid Anhydride The results of ozonolyses of enol ethers from 1,2‐ and 1,3‐dicarbonyl compounds presented here strongly indicate that these reactions do not proceed via the established Criegee ozonolysis mechanism for nucleophilic CC bonds. The quantitative one‐step synthesis of phthalonic acid anhydride via ozonolysis of 2‐(methoxymethyliden)‐1H‐inden‐1,3(2H)‐dione (28a) is described. Furthermore, a revision of the theory of alkene ozonolysis in the presence of tetracyanoethylene (TCNE) is proposed on the basis of a single‐electron‐transfer (SET) chemistry.
Isolation and Structure Elucidation of Enniatins L, M1, M2, and N: Novel Hydroxy Analogs
Authors: Vongvilai Pornrapee , Isaka Masahiko , Kittakoop Prasat , Srikitikulchai Prasert , Kongsaeree Palangpon , Prabpai Samran , Thebtaranonth Yodhathai
Published: August 2004

Four new cyclohexadepsipeptides, enniatins L (1), M1 (2), M2 (3), and N (4), have been isolated from an unidentified fungus (BCC 2629), together with the known enniatins B (5), H (6), and I (7), MK1688 (8), and enniatin B4 (9). Compounds 1–4 are the first enniatin analogs with an OH group at the side chain of one of the 2‐hydroxycarboxylic acid residues. The structures of 1–4 were elucidated by spectroscopic means and by X‐ray crystallography.
A New Preparation of 1,3,3‐Trimethylbicyclo[2.2.2]octan‐2,6‐dione, a Never Isolated Intermediate in a Total Synthesis of (+)‐Norpatchoulenol. Formal Total Synthesis of (±)‐Iso‐Norpatchoulenol
Authors: La Bella Angela , Leonelli Francesca , De Salve Ilse , Migneco Luisa M. , Marini Bettolo Rinaldo
Published: August 2004

A new preparation and the isolation and spectroscopic characterization of 1,3,3‐trimethylbicyclo[2.2.2]octan‐2,6‐dione (3), a so far elusive key intermediate in the Liu–Ralitsch total synthesis of (+)‐norpatchoulenol ((+)‐1a), is described. The preparation of 3 constitutes also a formal total synthesis of (±)‐iso‐norpatchoulenol ((±)‐1b), since 3 is correlated to an intermediate in the Monti and co‐workers synthesis of (±)‐1b.
Acovulparine, a New Norditerpene Alkaloid from Aconitum vulparia
Authors: Csupor Dezső , Forgo Peter , Máthé Imre , Hohmann Judit
Published: August 2004

Acovulparine (=1α,6β,7R,8β,14α,16β)‐4‐(hydroxymethyl)‐1,6,14,16‐tetramethoxyaconit‐19‐ene‐7,8‐diol; 1), a new norditerpene alkaloid, was isolated from the MeOH extract of the whole plants of Aconitum vulparia Reichenb., together with the known compounds lycoctonine (2) and delcosine (3). The structures were established by HR‐EI‐MS and advanced 2D‐NMR, including 1H‐NMR, JMOD, 1H,1H‐COSY, HSQC, and HMBC experiments. Acovulparine was found to contain the rare C(19)N azomethine group.
Confirmation of the Absolute (3R,3′S,6′R)‐Configuration of (all‐E)‐3′‐Epilutein
Authors: Molnár Péter , Deli József , Ősz Erzsébet , Zsila Ferenc , Simonyi Miklós , Tóth Gyula
Published: August 2004

Circular dichroism (CD) spectroscopy was used to distinguish between the isomeric (all‐E)‐configured 3′‐epilutein (2) and 6′‐epilutein (8) to establish the absolute configuration of epilutein samples of different (natural and semisynthetic) origin, including samples of 2 obtained from thermally processed sorrel. Thus, the CD data of lutein (1) and epilutein samples (2) were compared. Our results unambiguously confirmed the (3R,3′S,6′R)‐configuration of all epilutein samples. Compound 2 was thoroughly characterized, and its 13C‐NMR data are published herewith for the first time.
(E/Z)‐Isomerization of 3′‐Epilutein
Authors: Molnár Péter , Deli József , Ősz Erzsébet , Matus Zoltán , Tóth Gyula , Zsila Ferenc
Published: August 2004

3′‐Epilutein (=(all‐E,3R,3′S,6′R)‐4′,5′‐didehydro‐5′,6′‐dihydro‐β,β‐carotene‐3,3′‐diol; 1), isolated from the flowers of Caltha palustris, was submitted to both thermal isomerization and I2‐catalyzed photoisomerization. The structures of the main products (9Z)‐1, (9′Z)‐1, (13Z)‐1, (13′Z)‐1, (15Z)‐1, and (9Z,9′Z)‐1 were determined based on UV/VIS, CD, 1H‐NMR, and MS data.
Variation in the Settling Velocity of Suspended Particulate Matter in Shallow Lakes, with Special Implications for Mass Balance Modelling
Authors: Malmaeus Jan Mikael
Published: August 2004

Measurements of sedimentation were combined with water samples to calculate settling velocity of suspended particulate matter (SPM) in lakes. The study sites were open water stations and enclosures in Lake Erken (Sweden) and Lake Balaton (Hungary). Settling velocities were found to vary considerably both inside and outside the enclosures. Within sites, the differences between 25th and 75th percentiles of measured settling velocities of SPM were two‐ or three‐fold. Median settling velocities of SPM ranged from around 0.5 m/d in the enclosures of Lake Erken to more than 8 m/d in the open water of Lake Balaton. Special relevance was attributed to flocculation, which is known to be affected by, e.g., SPM concentration and turbulence. Even though not directly measured, the less turbulent environment inside the enclosures was suggested to explain the low settling velocity compared to the open water environment. Settling velocity apparently correlated with water current speed (r2 = 0.66; n = 12). Stepwise multiple linear regressions were used to relate the variability in settling velocity of SPM to the variability of possible controlling factors in a number of data subsets. In most cases, one variable describing the total amount of settling material (e.g., SPM) and one variable reflecting the composition of settling material (e.g., total phosphorus) were chosen. The use of suspended solids concentration to predict settling velocity in mass balance models was discussed. It was found that the mean slope between SPM and vSPM was close to 0.1 (m4d–1 g–1). (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Epithelium and/or theca are required for ATP‐elicited K+ current in follicle‐enclosed Xenopus oocytes
Authors: Saldaña Carlos , Vázquez‐Cuevas Francisco , Garay Edith , Arellano Rogelio O.
Published: August 2004

The Xenopus follicular cell membrane is endowed with ATP‐sensitive K+ channels, which are operated by various transmitters. These generate the ionic response named IK,cAMP via a mechanism that involves intracellular cAMP synthesis. It is known that opening these K+ channels favors oocyte maturation. Follicle stimulation by adenosine (Ado) or ATP consistently generates a strong IK,cAMP via activation of P1 and P3 purinergic receptors; however, ATP can also inhibit IK,cAMP, apparently acting on a third receptor type. Here, we show that IK,cAMP might be elicited by ATP released within the follicle, and that current activation by ATP was entirely dependent on the presence of epithelial and/or theca layers. Morphological studies confirmed that removal of epithelium/theca in these follicles (e.t.r.) was complete, and activation of fast Cl− (Fin) currents by ATP in e.t.r. follicles confirmed that communication between oocyte and follicular cells remained unchanged. Thus, dependence on epithelium/theca was specific for ATP‐elicited K+ current. Using UTP and βγ‐MeATP as specific purinergic agents for IK,cAMP inhibition and activation, respectively, it was found that inhibition of IK,cAMP elicited by ATP or UTP was robustly present in e.t.r. follicles but was absent or strongly decreased in whole follicles (w.f.). Accordingly, this indicated that in w.f., epithelium and/or theca downregulated the IK,cAMP inhibition evoked by ATP, and that this control mechanism was absent in e.t.r. follicles. We suggest that this notable action on follicular cells involves one or both of two mechanisms, a paracrine transmitter released from epithelial and/or theca layers and action of ecto‐ATPases. © 2004 Wiley‐Liss, Inc.
Differential expression of claudin‐2 along the human intestine: Implication of GATA‐4 in the maintenance of claudin‐2 in differentiating cells
Authors: Escaffit F. , Boudreau F. , Beaulieu J.‐F.
Published: August 2004

Claudins, and particularly claudin‐2, are important regulatory components of tight junction permeability. A better understanding of the involvement of claudin‐2 in intestinal barrier functions requires the characterization of its distribution and regulation in the intestine. Interestingly, the claudin‐2 gene promoter harbors a number of similarities to that of sucrase‐isomaltase, a marker of enterocyte differentiation. We thus investigated the expression of claudin‐2 in relation to the transcription factors CDX2, HNF‐1α, and GATA‐4 in the human intestine. The characterization of claudin‐2 and the expression of the above transcription factors were performed by immunofluorescence, Western blot, and RT‐PCR in the developing human intestinal epithelium. The functional role of CDX2, HNF‐1α, and GATA‐4 on claudin‐2 regulation was also examined by ectopic expression studies in intestinal cell models. Claudin‐2 was detected in both crypt and villus cells of the small intestine but restricted to undifferentiated crypt cells in the colon. CDX2 and HNF‐1α were expressed along the entire intestine whereas GATA‐4 was undetectable in the colon. Accordingly, in the colonic Caco‐2 cell model, claudin‐2 was found to be present only in undifferentiated cells. Like in the colonic epithelium, GATA‐4 was found to be also lacking in Caco‐2 cells while CDX2 and HNF‐1α were present at significant levels. Cotransfection experiments showed that the claudin‐2 promoter was activated by CDX2, HNF‐1α, and GATA‐4 in a cooperative manner. Furthermore, forced GATA‐4 expression in Caco‐2 cells enhances maintenance of claudin‐2 expression during differentiation. These observations suggest that optimal claudin‐2 expression in the gut relies on the presence of GATA‐4, suggesting a role for this factor in intestinal regionalization. © 2004 Wiley‐Liss, Inc.
Effect of tumor‐associated mutant E‐cadherin variants with defects in exons 8 or 9 on matrix metalloproteinase 3
Authors: Fuchs Margit , Hermannstädter Christine , Specht Katja , Knyazev Pjotr , Ullrich Axel , Rosivatz Erika , Busch Raymonde , Hutzler Peter , Höfler Heinz , Luber Birgit
Published: August 2004

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. The cell adhesion molecule E‐cadherin is frequently down‐regulated or mutated in tumors. In addition to down‐regulation of cell adhesion, degradation of the extracellular matrix by matrix metalloproteinases is necessary for tumor cell spread. To investigate a possible link between E‐cadherin and matrix metalloproteinase 3 (MMP‐3), we examined expression of MMP‐3 in human MDA‐MB‐435S cells transfected with wild‐type (wt) or three different tumor‐associated mutant E‐cadherin variants with alterations in exons 8 or 9, originally identified in gastric carcinoma patients. In the presence of wt E‐cadherin, the MMP‐3 protein level was decreased in cellular lysates and in the supernatant where a secreted form of the protein is detectable. Down‐regulation of MMP‐3 was not found in MDA‐MB‐435S transfectants expressing mutant E‐cadherin variants which indicates that E‐cadherin mutations interfere with the MMP‐3 suppressing function of E‐cadherin. The mechanism of regulation of MMP‐3 by E‐cadherin is presently not clear. We have previously found that cell motility is enhanced by expression of the mutant E‐cadherin variants used in this study. Here, we found that application of the synthetic inhibitor of MMP‐3 NNGH and small interfering RNA (siRNA) directed against MMP‐3 reduce mutant E‐cadherin‐enhanced cell motility. Taken together, our results point to a functional link between MMP‐3 and E‐cadherin. MMP‐3 is differentially regulated by expression of wt or mutant E‐cadherin. On the other hand, MMP‐3 plays a role in the enhancement of cell motility by mutant E‐cadherin. Both observations may be highly relevant for tumor progression since they concern degradation of the extracellular matrix and tumor cell spread. © 2004 Wiley‐Liss, Inc.
Sugarcane bagasse hydrolysis with phosphoric and sulfuric acids and hydrolysate detoxification for xylitol production
Authors: Carvalho Walter , Batista Marcio A , Canilha Larissa , Santos Julio C , Converti Attilio , Silva Silvio S
Published: August 2004

The effectiveness of phosphoric acid to release xylose from sugarcane bagasse hemicellulose was assessed through a 23 full factorial design. The maximum xylose concentration in the hydrolysate (17.1 g dm−3) was attained when the bagasse was treated at 160 °C for 60 min, using 70 mg of phosphoric acid per gram of dry‐bagasse. Hydrolysis carried out with sulfuric acid, under optimum conditions previously determined, provided a hydrolysate with a similar xylose concentration (17.2 g dm−3). After vacuum concentration, these hydrolysates were detoxified and used for xylitol production with the yeast Candida guilliermondii. Two different detoxification strategies, which consisted of adjusting the pH of the hydrolysates to 5.5 with either calcium oxide or ammonium hydroxide, both followed by active charcoal adsorption, were tested. The best xylitol productions (18.1 and 19.2 g dm−3) were observed when calcium oxide was used to adjust the pH of both the phosphoric and the sulfuric acid hydrolysates, respectively. Copyright © 2004 Society of Chemical Industry
Contrasting modes of natural selection acting on pigmentation genes in the Drosophila dunni subgroup
Authors: Wilder J.A. , Dyreson E.G. , O'Neill R.J. , Spangler M.L. , Gupta R. , Wilder A.S. , Hollocher H.
Published: August 2004

Genes that encode for divergent adaptive traits may have genealogies that contrast with those from loci that are not functionally involved in differentiation. Here, we examine DNA sequence variation among the species of the eastern Caribbean Drosophila dunni subgroup at two loci, yellow and dopa decaboxylase (Ddc), which both play integral roles in pigmentation patterning of adult Drosophila. Phylogenetic analyses of these loci produce gene genealogies with topologies that mirror those described for other nuclear genes: the six morphologically distinct species within the subgroup are divided into only three lineages, with one lineage containing four species that share extensive ancestral polymorphism. At the Ddc locus these major lineages are delineated only by silent site variation. We observe a significantly higher rate of synonymous site divergence than non‐synonymous divergence, consistent with strong purifying selection acting on the locus. In contrast, the yellow locus exhibits patterns of amino acid divergence and nucleotide diversity that are consistent with recent diversifying selection acting in two different lineages. This selection appears to be targeting amino acid variants in the signal sequence of the Yellow protein, a region which is tightly constrained among members of the larger D. cardini radiation. This result highlights not only the potential importance of yellow in the evolution of divergent pigmentation patterns among members of the D. dunni subgroup, but also hints that variation in signal peptide sequences may play a role in phenotypic diversification. J. Exp. Zool. (Mol. Dev. Evol.) 302B:1–14, 2004. © 2004 Wiley‐Liss, Inc.
Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells
Authors: Sakuragawa Norio , Kakinuma Kenichi , Kikuchi Aiko , Okano Hideyuki , Uchida Saiko , Kamo Isao , Kobayashi Mamoru , Yokoyama Yasunobu
Published: August 2004

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin‐positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66–82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT‐PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti‐Tuj1 and NF‐M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein‐positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC‐derived stem cells may be a favorable cell vehicle in regenerative medicine. © 2004 Wiley‐Liss, Inc.

An Erratum has been published for this article in J Neurosci Res 79: 725, 2005.
Hormonal control of somatic cell oocyte interactions during ovarian follicle development
Authors: Combelles Catherine M.H. , Carabatsos Mary Jo , Kumar T. Rajendra , Matzuk Martin M. , Albertini David F.
Published: August 2004

In the mammalian ovarian follicle, paracrine signaling between the oocyte and somatic granulosa cells is bidirectional but the structural basis and physiological regulations of communication between gametic and somatic compartments remain unknown. The present experiments were designed to test the hypothesis that follicle stimulating hormone (FSH) regulates the ability of granulosa cells to make connections with the oocyte. We show that in prepubertal unprimed mice and mice carrying a targeted deletion of the FSHβ subunit gene, granulosa cells exhibit orientation towards the oocyte manifest by the elaboration of transzonal projections (TZPs) and “apical” centrosome positioning at sites of granulosa‐zona contact. In vivo FSH treatment results in a retraction of TZPs. Coincident with TZP retraction induced by FSH are changes in oocyte transcriptional activity and meiotic competence, which suggests one means by which the oocyte‐granulosa cell dialogue may be modulated during development of ovarian follicles. Mol. Reprod. Dev. 69: 347–355, 2004. © 2004 Wiley‐Liss, Inc.
Birth of calves expressing the enhanced green fluorescent protein after transfer of fresh or vitrified/thawed blastocysts produced by somatic cell nuclear transfer
Authors: Gong Guochun , Dai Yunping , Fan Baoliang , Zhu Huabing , Zhu Shien , Wang Haiping , Wang Lili , Tang Bo , Li Rong , Wan Rong , Liu Ying , Huang Yinhua , Zhang Lei , Sun Xiuzhu , Li Ning
Published: August 2004

The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE‐EGFP‐IRES‐NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin‐resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum‐fed (73.6 and 47.2%, respectively) or serum‐starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP‐embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully. Mol. Reprod. Dev. 69: 278–288, 2004. © 2004 Wiley‐Liss, Inc.
Macaque sperm release ESP13.2 and PSP94 during capacitation: The absence of ESP13.2 is linked to sperm‐zona recognition and binding
Authors: Tollner Theodore L. , Yudin Ashley I. , Treece Cathy A. , Overstreet James W. , Cherr Gary N.
Published: August 2004

ESP13.2 coats the entire surface of macaque sperm and remains until sperm become capacitated (Yudin et al., 2003: Biol Reprod 69: 1118–1128). Capacitation of macaque sperm is synchronized by treatment with dibutyrl cAMP (dbcAMP) and caffeine. ESP13.2 and PSP94 constituted ∼95% of the proteins released from the sperm surface following treatment with caffeine + dbcAMP. Caffeine and dbcAMP alone induce different patterns of ESP13.2 release. As determined by ELISAs of supernatants and immuno‐fluorescent labeling of sperm heads, caffeine alone and caffeine + dbcAMP induced comparable release of ESP13.2, while dbcAMP‐treated sperm did not differ from controls. Sperm treated with caffeine + dbcAMP showed a reduction of ESP13.2 from the entire surface, while caffeine treatment alone induced removal of ESP13.2 from the sperm head and midpiece. As confirmed with immunofluorescence, ESP13.2 could be added back to the surfaces of sperm that had been previously exposed to caffeine. Treatment with caffeine significantly increased the number of sperm that bound tightly to the zona pellucida as compared with controls (42 ± 9 and 13 ± 3 sperm/zona, respectively; P ≤ 0.01). This increase in binding was inhibited by “adding back” ESP13.2 to the sperm surface (12.8 ± 3; P ≤ 0.01). Alexa‐conjugated anti‐ESP13.2 Ig labeling of live sperm showed that only sperm lacking ESP13.2 over the head were capable of tight binding to the zona. Our results suggest that ESP13.2 masks zona pellucida ligands on the sperm surface and its release, as part of capacitation, is required for sperm–zona interaction. Mol. Reprod. Dev. 69: 325–337, 2004. © 2004 Wiley‐Liss, Inc.
Regulation of cleavage by protein kinase C in Chaetopterus
Authors: Yang Dazhi , Hinton Shantá D. , Eckberg William R.
Published: August 2004

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC‐γ transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC‐α and ‐β transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC‐γ activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC‐α and/or ‐β from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down‐regulation of this activity is essential for cleavage after fertilization. Mol. Reprod. Dev. 69: 308–315, 2004. © 2004 Wiley‐Liss, Inc.
Molecular modification of Penaeus monodon sperm in female thelycum and its consequent responses
Authors: Vanichviriyakit Rapeepun , Kruevaisayawan Hathairat , Weerachatyanukul Wattana , Tawipreeda Pattira , Withyachumnarnkul Boonsirm , Pratoomchat Boonyarath , Chavadej Jittipan , Sobhon Prasert
Published: August 2004

Using Penaeus monodon as the model, we demonstrated the molecular changes and the mechanism of thelycal‐dependent sperm modification resulting in an enhanced acrosome reaction (AR) response. Attention was paid to the modification of the sperm plasma membrane which was mediated through an adsorption or removal of sperm peripheral and integral membrane proteins as indicated by the different profiles of these proteins in spermatophore (S) and thelycal (T) sperm. In vitro adsorption of Alexa‐488 conjugated T proteins onto the entire S‐sperm surface confirmed protein transfer in a time–dependent manner. Specific anchoring of 83 and 140 kDa proteins to sperm peripheral proteins as well as 53/55 and 60 kDa proteins to sperm lipids was demonstrated. Apart from membrane modification, a substantial increase in protein tyrosine phosphorylation was shown to be closely associated with T‐dependent sperm modification event. The physiological significance of this sperm modification in enhancing sperm AR response, which required at least 3 days of T residence in order for the sperm to gain a complete AR response, was also elucidated. Mol. Reprod. Dev. 69: 356–363, 2004. © 2004 Wiley‐Liss, Inc.
Expression of HSP70/HSC70 in swine blastocysts: Effects of oxidative and thermal stress
Authors: Bernardini Chiara , Fantinati Paolo , Zannoni Augusta , Forni Monica , Tamanini Carlo , Bacci Maria Laura
Published: August 2004

Stress proteins are induced in response to a wide range of biological and physicochemical stresses; HSC70 (constitutive) and HSP70 (inducible) belong to the HSP70 stress protein family. The present study aimed at investigating whether environmental stress, particularly thermal and oxidative stress, is involved in modulating HSP70/HSC70 expression in in vitro porcine embryos from two/four cell stage to blastocyst. For oxidative stress, embryos were cultured at 38.5°C under a 5% O2–5% CO2 atmosphere or 5% CO2 in air (approximately 20% O2); for thermal stress, embryos were cultured at 38.5°C under 5% O2, 5% CO2, and exposed to heat shock (1 hr at 42°C). At the end of culture, embryos were analysed by Western blotting, using specific antibodies discriminating HSP70 from HSC70. Embryos cultured under 20% O2 showed HSC70 levels significantly higher (P < 0.005) than embryos cultured under 5% O2, while heat shocked embryos presented HSP70 levels higher (P < 0.01) than control group. In addition, the developmental rate of embryos was negatively affected by the higher oxygen tension (P < 0.05). Our data indicate that porcine embryos express both HSP70 and HSC70 and could differentially respond to both oxidative and heat stress by up‐regulating HSC70 and HSP70, respectively. Mol. Reprod. Dev. 69: 303–307, 2004. © 2004 Wiley‐Liss, Inc.
Transcription factor sox‐2 inhibits co‐activator stimulated transcription
Authors: Bernadt Cory T. , Nowling Tamara , Rizzino Angie
Published: August 2004

Previous studies have shown that transcription of the fibroblast growth factor‐4 (FGF‐4) gene by early embryonic cells is dependent upon a powerful distal enhancer located 3 kb downstream of the transcription start site within the untranslated region of the last exon. The transcription factors Sox‐2 and Oct‐3 cooperatively bind to critical cis‐regulatory elements within the enhancer to synergistically activate transcription. Moreover, the co‐activator p300 can mediate the synergistic activity of Sox‐2 and Oct‐3, and p300 associates with the FGF‐4 enhancer in vivo. Embryonal carcinoma (EC) cells have been used extensively as a model system to study the regulation of the FGF‐4 gene during early development. Recently, it has been suggested that suboptimal levels of Sox‐2 expression in F9 EC cells limit the transcription of the FGF‐4 gene. The studies presented in this report argue that Sox‐2 levels are not limiting in F9 EC cells. Moreover, overexpression of Sox‐2 in F9 EC cells decreases FGF‐4 promoter activity. In addition, overexpression of Sox‐2 in these cells inhibits activation by the co‐activators p300, CBP, and OCA‐B in a manner that requires the transactivation domain of Sox‐2. These findings suggest that Sox‐2 levels in F9 EC cells are regulated carefully to avoid interference with the transcription of critical genes. Mol. Reprod. Dev. 69: 260–267, 2004. © 2004 Wiley‐Liss, Inc.
A mass spectrometric “Western blot” to evaluate the correlations between histone methylation and histone acetylation
Authors: Zhang Kangling , Siino Joseph S. , Jones Patrick R. , Yau Peter M. , Bradbury E. Morton
Published: August 2004

Histone acetylation, methylation, and phosphorylation occur predominantly in the unstructured N‐terminal domains or histone “tails”. These modifications and others comprise a “histone code” that directly facilitates or antagonizes association of regulatory proteins with nucleosomes to mediate changes in chromatin structure and activity. Methylation of histone H3 outside of the tail region at lysine 79 has been reported for a variety of species ranging from yeast to humans and in some gene‐specific cases appears to be associated with active chromatin and transcription. Whether methylation of lysine 79 is associated with other post‐translational modifications of the H3 tail is unknown. Using mass spectrometric relative quantitation, a mass spectrometric “Western blot”, we compare methylation at lysines 4, 9, and 79 with acetylation of human histone H3. We find that the total levels of lysine 4 and 79 methylation (combined mono‐, di‐, and trimethylation) in the H3 population increase with the degree of H3 tail acetylation. The total amount of lysine 4 methylation increases progressively from less than 10% in the nonacetylated H3 to greater than 90% in the penta‐acetylated H3. In addition, significant levels of lysine 4 trimethylation also occur in combination with the penta‐acetylated H3 species. In contrast, the level of H3 lysine 9 trimethylation is greatest for the monoacetylated species while H3 lysine 9 acetylation occurs predominantly in hyperacetylated (tetra‐ and penta‐acetylated) H3 isoforms. Together, these results indicate that methylation of lysine 4 and 79 as well as the switch from lysine 9 methylation to acetylation are coordinated synchronously with H3 hyperacetylation as marks of active chromatin.
Protein identification and tracking in two‐dimensional electrophoretic gels by minimal protein identifiers
Authors: Mattow Jens , Schmidt Frank , Höhenwarter Wolfgang , Siejak Frank , Schaible Ulrich E. , Kaufmann Stefan H.E.
Published: August 2004

Protein identification by matrix‐assisted laser desorption/ionization mass‐spectrometry peptide mass fingerprinting (MALDI‐MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low‐molecular‐mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI‐MS‐based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two‐dimensional gel electrophoresis (2‐DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI‐MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein‐specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF‐Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2‐DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.
Genome and proteome analysis of Chlamydia
Authors: Vandahl Brian B. S. , Birkelund Svend , Christiansen Gunna
Published: August 2004

It has been difficult to study the molecular biology of the obligate intracellular bacterium Chlamydia due to lack of genetic transformation systems. Therefore, genome sequencing has greatly expanded the information concerning the biology of these pathogens. Comparing the genomes of seven sequenced Chlamydia genomes has provided information of the common gene content and gene variation. In addition, the genome sequences have enabled global investigation of both transcript and protein content during the developmental cycle of chlamydiae. During this cycle Chlamydia alternates between an infectious extracellular form and an intracellular dividing form surrounded by a phagosome membrane termed the chlamydial inclusion. Proteins secreted from the chlamydial inclusion into the host cell may interact with host cell proteins and modify the host cell's response to infection. However, identification of such proteins has been difficult because the host cell cytoplasm of Chlamydia infected cells cannot be purified. This problem has been circumvented by comparative proteomics.