bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of the Apical Plasma Membrane Recycling System During Cell Division
Authors: Hobdy‐Henderson Karen C. , Hales Chadwick M. , Lapierre Lynne A. , Cheney Richard E. , Goldenring James R.
Published: September 2003

The members of the family of Rab11 small GTPases are critical regulators of the plasma membrane vesicle recycling system. While previous studies have determined that the Golgi apparatus disperses during mitosis and reorganizes after cytokinesis, the fate of the recycling system during the cell cycle is more obscure. We have now studied in MDCK cells the fate during mitosis of an apical recycling system cargo, the polymeric IgA receptor (pIgAR), and regulators of the recycling system, Rab11a and its interacting proteins myosin Vb, Rab11‐FIP1, Rab11‐FIP2 and pp75/Rip11. Rab11a, pIgAR and myosin Vb containing vesicles dispersed into diffuse puncta in the cytosol during prophase and then became clustered near the spindle poles after metaphase, increasing in intensity throughout telophase. A similar pattern was observed for Rab11‐FIP1 and Rab11‐FIP2. However, Rab11‐FIP1 lost colocalization with other recycling system markers during late prophase, relocating to the pericentriolar material. During telophase, Rab11‐FIP1 returned to recycling system vesicles. Western blot analysis indicated that both Rab11a and pIgAR remained associated with membrane vesicles throughout the cell cycle. This behavior of the Rab11a‐containing apical recycling endosome system during division was distinct from that of the Golgi apparatus. These results indicate that critical components of the apical recycling system remain associated on vesicles throughout the cell cycle and may provide a means for rapid re‐establishment of plasma membrane components after mitosis.
Increasing Circulating T‐cell Activation Markers are Linked to Subsequent Implantation Failure After Transfer of In vitro Fertilized Embryos
Authors: Coulam Carolyn B. , Roussev Roumen G.
Published: September 2003

Problem: Implantation determines success of in vitro fertilization (IVF) and embryo transfer (ET) cycles. Data are accumulating to support a role of the immune system in implantation. Most of the literature addresses the importance of natural killer (NK) cells in this process. The purpose of the current study is to examine the role of circulating T cells in implantation failure.

Method of study: Blood from 22 women undergoing IVF/ET during November, 2001, was drawn on cycle day 9 and analyzed for the percentage of circulating T cells expressing the activation markers CD69+ and human leukocyte antigen (HLA)‐DR and the suppressor marker CD11b using immunofluorescence and flow cytometry. These results were compared with total percentage circulating CD3, CD4 and CD8 cells as well as NK cells and pregnancy outcome that cycle.

Results: Infertile women had significantly greater expression of the activation marker of CD69+ among CD8+ and CD4+ T cells and HLA‐DR among CD4 cells than fertile women. No difference in expression of T cell suppressor marker of CD11b was noted when infertile and fertile women were compared. No correlations were observed when activated T cells were compared with circulating CD3+, CD4+, CD8+, activated NK cells and NK cytotoxicity. CD3+4+HLA‐DR+ was expressed significantly less among successfully pregnant compared with unsuccessfully pregnant women.

Conclusion: T‐cell activation markers CD 69+ and HLA‐DR+ are associated with increased implantation failure after IVF/ET.
Evidence for a Correlation between Trophoblast Invasiveness and STAT3 Activity
Authors: Corvinus Florian M. , Fitzgerald Justine S. , Friedrich Karlheinz , Markert Udo R.
Published: September 2003

Problem: Extravillous trophoblast cells are capable of invading decidual tissue during early pregnancy. This property is reminiscent of cancer cells. The invasiveness of trophoblasts, however, extends only to a well‐regulated limit. Signal transduction processes underlying this phenomenon are as yet poorly characterized. Many factors involved in trophoblast invasiveness are known to trigger intracellular signaling cascades in other cell types that ultimately lead to the activation of signal transducers and activators of transcription (STATs). STAT3 activity was recently found related to the malignant phenotype of different tumor cells and potentially contributes to their invasive properties.

Method of Study: We investigated the status of STAT3 activity in ex vivo trophoblast cells from first trimester and term placentae employing an electrophoretic mobility shift assay (EMSA) and compared it with that of a highly malignant choriocarcinoma cell line.

Results: Specific DNA binding activity of two STAT3 variants (STAT3α and β) was observed in immature trophoblasts and appeared to be lost in term placentae. The malignant phenotype of choriocarcinoma cells coincides with a high degree of STAT3 activity.

Conclusion: These results suggest a connection between STAT3 activity and trophoblast invasiveness.
Traumatic intrusion of maxillary permanent incisors into the nasal cavity associated with a seizure disorder: report of a case
Authors: Martin Brian S.
Published: September 2003

Abstract –  Childhood traumatic dental injury is typically associated with motor vehicle accidents, sports, or other recreational activities. However, seizure disorders or other compromising medical conditions may precipitate dental trauma. Complete intrusion injuries in the mixed or permanent dentition are relatively rare. This paper describes the case history of an adolescent male who sustained displacement of a lateral incisor into the nasal cavity and complete bony intrusion of the three other permanent maxillary incisors because of a fall following a seizure. Counseling on helmet/face‐shield usage for seizure‐susceptible patients must be provided and documented, particularly after sustaining traumatic injury.
A multicentre investigation into the role of structured histories for patients with tooth avulsion at their initial visit to a dental hospital
Authors: Day Peter F. , Duggal Monty S.
Published: September 2003

Abstract  – A paper structured history (SH) is a sheet, which prompts or reminds the clinician to ask various important questions. The aim of this study was to examine avulsion cases with respect to the quality of clinical records. Hospitals studied used either a paper SH or had no specific structure in their recording of avulsion details, e.g. unstructured histories (USH). The most important prognostic items that should be recorded for avulsion cases at their first visit were identified by reviewing the literature. Clinical case records meeting strict inclusion criteria were retrospectively analyzed against 10 important prognostic items. Forty‐seven patient records were identified in the SH group compared to 43 patient records in the USH group. Using chi‐square and Fisher's exact tests, the SH group were significantly better at recording the following: accident details (P < 0.001), loss of consciousness (P < 0.001), other teeth or tooth injuries (P < 0.05), extra‐alveolar mediums (P < 0.01), total extra‐alveolar time (P < 0.001), antibiotics given at time of injury (P < 0.05) and apical maturity (P < 0.001). In all the dental hospitals selected, two‐thirds of the case records were completed by junior dentists not in specialist training and the improvement in history when using an SH form was most pronounced in these groups. It is concluded, therefore, that an SH should be taken for cases of avulsion as it was significantly better at collecting essential prognostic information.
Root resorption in dental trauma: 45 cases followed for 5 years
Authors: Majorana A. , Bardellini E. , Conti G. , Keller E. , Pasini S.
Published: September 2003

Abstract –  We aimed to: (i) study the prevalence of root resorption after luxation or avulsion injuries on permanent teeth referred to our dental clinic over a 3‐year period; (ii) study the relationship between type of injury and resultant type of resorption complication; and (iii) evaluate success of treatment protocols for various resorption complications. We observed 1943 patients with dental trauma, aged between 2 and 26 years, referred to the Accident and Emergency Department of the Dental Clinic, University of Brescia, from 1st September 1997 to 31st December 2001. Of these, 261 permanent teeth had sustained luxation (n = 188) or avulsion (n = 73) injuries. Permanent teeth luxation and avulsion injuries occurred most often in upper incisors (75%) of patients mostly aged 12–21 years, with males more commonly affected than females (68.3% vs. 31.7%). These cases were followed for 5 years, and complications and response to treatment were recorded. Root resorption was observed in 45 (17.24%) of these cases. Of the 45 cases with resorption, 9 were associated with luxation injury (20%) while 36 (80%) with avulsion. We distinguished 30 cases of inflammatory root resorption (18 transient and 12 progressive) and 15 cases of ankylosis and osseous replacement. When resorption was recognized, quick and effective treatment could still result in an excellent functional and aesthetic outcome for these teeth.
Clinical outcomes for permanent incisor luxations in a pediatric population. III. Lateral luxations
Authors: Nikoui Mandana , Kenny David J. , Barrett Edward J.
Published: September 2003

Abstract –  A longitudinal outcome study was designed to identify variables that influenced tooth survival as well as pulpal and periodontal outcomes of laterally luxated permanent maxillary incisors of children and adolescents. All cases were treated between June 1988 and June 1998 in a teaching hospital clinic. Clinical and radiographic data were collected for 42 patients (26 males, 16 females) that represented 58 permanent maxillary incisors. Mean age at the time of injury was 11.4 years (range: 6.3–17.8 years). Mean follow‐up time was 1460 days (range: 183–3905 days). In the entire sample (n = 58), no incisors required extraction. Survival analysis and logistic regression were used to identify variables significantly related to the survival and healing outcomes of these incisors. Pulp necrosis (PN) (40%) and pulp canal obliteration (PCO) (40%) were common healing complications. Proportional hazards (Cox) regression showed that PN develops within the first year. Logistic regression demonstrated that root development (P = 0.3, PN; P = 0.8, PCO) and extent of lateral luxation (P = 0.5, PN; P = 0.9, PCO) were not significantly related to PN and PCO. This study provides the first report of incisor survival in children and adolescents following lateral luxation injuries.
5,10-Methenyltetrahydrofolate cyclohydrolase, rat liver and chemically catalysed formation of 5-formyltetrahydrofolate
Authors: BAGGOTT Joseph E. , MacKENZIE Robert E.
Published: September 2003

The 5,10-methenyltetrahydrofolate (5,10-CH=H4folate) synthetase catalyses the physiologically irreversible formation of 5,10-CH=H4folate from 5-formyltetrahydrofolate (5-HCO-H4folate) and ATP. It is not clear how (or if) 5-HCO-H4folate is formed in vivo. Using a spectrophotometric assay for 5-HCO-H4folate, human recombinant 5,10-CH=H4folate cyclohydrolase, which catalyses the hydrolysis of 5,10-CH=H4folate to 10-HCO-H4folate, was previously shown to catalyse inefficiently the formation of 5-HCO-H4folate at pH 7.3 [Pelletier and MacKenzie (1996) Bioorg. Chem. 24, 220–228]. In the present study, we report that (i) the human cyclohydrolase enzyme catalyses the conversion of 10-HCO-/5,10-CH=H4folate into 5-HCO-H4folate (it is also chemically formed) at pH 4.0–7.0; (ii) rat liver has a very low capacity to catalyse the formation of 5-HCO-H4folate when compared with the traditional activity of 5,10-CH=H4folate cyclohydrolase and the activity of the 5,10-CH=H4folate synthetase; and (iii) a substantial amount of 5-HCO-H4folate reported to be present in rat liver is chemically formed during analytical procedures. We conclude that (i) the cyclohydrolase represents some of the capacity of rat liver to catalyse the formation of 5-HCO-H4folate; (ii) the amount of 5-HCO-H4folate reported to be present in rat liver is overestimated (liver 5-HCO-H4folate content may be negligible); and (iii) there is little evidence that 5-HCO-H4folate inhibits one-carbon metabolism in mammals.
Modulation of DNA topoisomerase IIα promoter activity by members of the Sp (specificity protein) and NF-Y (nuclear factor Y) families of transcription factors
Authors: MAGAN Natisha , SZREMSKA Agnieszka P. , ISAACS Richard J. , STOWELL Kathryn M.
Published: September 2003

topo IIα (topoisomerase IIα) is a major target of several commonly used anticancer drugs and is subject to down-regulation at the transcriptional level in some drug-resistant cell lines and tumours in response to chemotherapy. Clinical resistance to such drugs has been correlated with down-regulation of topo IIα at transcription in some drug-resistant cell lines and tumours. Putative binding sites for a variety of transcription factors, including Sp1 (specificity protein 1) and NF-Y (nuclear factor Y) have previously been identified in the topo IIα promoter, but their functional significance and interactions have not been described following exposure to anti-cancer drugs. The binding of these factors to specific putative regulatory elements in the topo IIα promoter was studied using electrophoretic-mobility-shift assays. Sp1 was found to bind strongly to both distal and proximal GC-rich elements and NF-Y to ICB1 (the first inverted CCAAT box). The functional significance of transcription-factor binding was studied using transient transfection of HeLa cells using a luciferase reporter driven by a 617-bp minimal promoter containing point mutations in putative regulatory elements. Sp1 and NF-Y were both found to be transcriptional modulators with activator or repressor functions depending on protein/DNA context. Moreover, a functional interaction between Sp1 and NF-Y bound at proximal elements was observed.
The directionality of chitin biosynthesis: a revisit
Authors: IMAI Tomoya , WATANABE Takeshi , YUI Toshifumi , SUGIYAMA Junji
Published: September 2003

The molecular directionality of chitin biosynthesis was investigated by transmission electron microscopy (TEM) using electron crystallography methods applied to reducing-end-labelled β-chitin microcrystals from vestimentiferan Lamellibrachia satsuma tubes and nascent β-chitin microfibrils from the diatom Thalassiosira weissflogii. The data allowed confirmation that the microfibrils were extruded with their reducing end away from the biosynthetic loci, an orientation consistent only with elongation through polymerization at the non-reducing end of the growing chains. Such a chain-extension mechanism, which has also been demonstrated for cellulose and hyaluronan, appears to be general for glycosyltransferases that belong to the GT2 (glycosyl transferase 2) family. The data also allowed confirmation that in β-chitin the chains are crystallized in a ‘parallel-up’ mode, in contrast with hypotheses proposed in previous reports.
Different modes of internalization of apoptotic alkyl-lysophospholipid and cell-rescuing lysophosphatidylcholine
Authors: van der LUIT Arnold H. , BUDDE Marianne , VERHEIJ Marcel , van BLITTERSWIJK Wim J.
Published: September 2003

The synthetic alkyl-lysophospholipid (ALP), Et-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine), can induce apoptosis in tumour cells. Unlike conventional chemotherapeutic drugs, ALP acts at the cell-membrane level. We have reported previously that ALP is internalized, and interferes with phosphatidylcholine (PC) biosynthesis de novo, which appeared to be essential for survival in lymphoma cells [Van der Luit, Budde, Ruurs, Verheij and Van Blitterswijk (2002) J. Biol. Chem. 277, 39541–39547]. Here, we report that, in HeLa cells, ALP accumulates in lipid rafts, and that internalization is inhibited by low temperature, monensin, disruption of lipid rafts and expression of a dominant-negative mutant of dynamin bearing a replacement of Lys44 with alanine (K44A). Thus ALP is internalized via raft- and dynamin-mediated endocytosis. Dynamin-K44A alleviated the ALP-induced inhibition of PC synthesis and rescued the cells from apoptosis induction. Additional cell rescue was attained by exogenous lysoPC, which after internalization serves as an alternative substrate for PC synthesis (through acylation). Unlike ALP, and despite the high structural similarity to ALP, lysoPC uptake did not occur via lipid rafts and did not depend on functional dynamin, indicating no involvement of endocytosis. Albumin back-extraction experiments suggested that (radiolabelled) lysoPC undergoes transbilayer movement (flipping). We conclude that ALP is internalized by endocytosis via lipid rafts to cause apoptosis, while exogenous cell-rescuing lysoPC traverses the plasma membrane outside rafts by flipping. Additionally, our data imply the importance of ether bonds in lyso-phospholipids, such as in ALP, for partitioning in lipid rafts.
A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi
Authors: GONZÁLEZ Jorge , CORNEJO Alberto , SANTOS Marcia R. M. , CORDERO Esteban M. , GUTIÉRREZ Bessy , PORCILE Patricio , MORTARA Renato A. , SAGUA Hernán , da SILVEIRA José Franco , ARAYA Jorge E.
Published: September 2003

Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 μM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin–Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.
Characterization of the interactions between Asp141 and Phe236 in the Mn2+–l-malate binding of pigeon liver malic enzyme
Authors: CHEN Yen-I , CHEN Yu-Hou , CHOU Wei-Yuan , CHANG Gu-Gang
Published: September 2003

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+–ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141→Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (ΔΔGint) between any two residues. The dissociation constants for Mn2+ (Kd,Mn) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the Kd,Mn for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of −2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.
Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases
Authors: KARANTZENI Irene , RUIZ Carmen , LIU Chin-Chi , LiCATA Vince J.
Published: September 2003

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5′ nuclease domain. Removal of the 5′ nuclease domains produces the ‘large fragment’ domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature Tm of 37 and 100 °C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5′ nuclease domains of each melting separately. The 5′ nuclease domain of Taq denatures as a separate peak, 10 °C before the Klentaq domain. Melting of the 5′ nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5′ nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower Tm values, but almost identical van't Hoff enthalpy ΔH values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.
Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide
Authors: ROMAN Elisabet , ROBERTS Ian , LIDHOLT Kerstin , KUSCHE-GULLBERG Marion
Published: September 2003

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAβ1,4-GlcNAcα1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-d-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.
Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells
Authors: YAMAGUCHI Hironori , KITAYAMA Joji , TAKUWA Noriko , ARIKAWA Kayo , INOKI Isao , TAKEHARA Kazuhiko , NAGAWA Hirokazu , TAKUWA Yoh
Published: September 2003

We have recently reported that S1P (sphingosine-1-phosphate) differentially regulates cellular Rac activity and cell migration in either a positive or a negative direction via distinct G-protein-coupled receptor subtypes, i.e. S1P1/Edg1 (endothelial differentiation gene) and S1P2/Edg5 respectively, when each of the S1P receptor subtypes is expressed in CHO (Chinese-hamster ovary) cells. In B16F10 mouse melanoma cells, in which S1P2, but not the other S1P receptor subtypes, is endogenously expressed, S1P inhibited cell migration with concomitant inhibition of Rac and stimulation of RhoA in dose-dependent manners. Overexpression of S1P2 in the melanoma cells resulted in potentiation of S1P inhibition of both Rac and cell migration. In contrast, overexpression of S1P1 led to stimulation of cell migration, particularly at the lower S1P concentrations. Treatment of B16F10 cells with S1P inhibited lung metastasis 3 weeks after injection into mouse tail veins. Intriguingly, overexpression of S1P2 greatly potentiated the inhibition of metastasis by S1P, whereas that of S1P1 resulted in aggravation of metastasis. Suppression of cellular Rac activity by adenovirus-transduced expression of N17Rac, but not N19RhoA, strongly inhibited cell migration in vitro and lung metastasis in vivo. These results provide the first evidence that G-protein-coupled receptors could participate in the regulation of metastasis, in which ligand-dependent, subtype-specific regulation of the cellular Rac activity is probably critically involved as a mechanism.
Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid
Authors: AITKEN Jacqueline F. , LOOMES Kerry M. , KONARKOWSKA Barbara , COOPER Garth J. S.
Published: September 2003

There is a significant correlation between the occurrence of pancreatic islet amyloid and β-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.
Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization
Authors: CHIU Sung-Kay , HSU Mandy , KU Wei-Chi , TU Ching-Yu , TSENG Yu-Tien , LAU Wai-Kwan , YAN Rong-Yih , MA Jing-Tyan , TZENG Chi-Meng
Published: September 2003

Most microarray slides are manufactured or coated with a layer of poly(l-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.
Oxidation of thioanisole and p-methoxythioanisole by lignin peroxidase: kinetic evidence of a direct reaction between compound II and a radical cation
Authors: BRÜCK Thomas B. , GERINI Maria Francesca , BACIOCCHI Enrico , HARVEY Patricia J.
Published: September 2003

The reaction of H2O2 with thioanisole and p-methoxythioanisole catalysed by lignin peroxidase from Phanerochaete chrysosporium has been studied spectrophotometrically under turnover and single turnover conditions with a stopped-flow apparatus. Pre-formed lignin peroxidase compounds I and II are each able to react with the sulphides to form a sulphide radical cation. The radical cation is then converted into the sulphoxide either by reaction with the medium or by reaction with compound II. This is the first report of a direct reaction between compound II and the substrate radical cation. With thioanisole, significant enantiomeric selectivity and high oxygen incorporation in the sulphoxide are obtained because compound II is preferentially reduced by the enzyme-bound thioanisole radical cation compared with the neutral substrate. By contrast, with p-methoxythioanisole, the data imply formation of an intermediate ternary complex comprising compound II, radical cation and neutral substrate, such that a chain of electron transfer reactions starting from neutral molecule and progressing to oxidized haem via substrate radical cation is facilitated, yielding the native enzyme and two molecules of p-methoxythioanisole radical cation as products. The reactions of compounds I and II with sulphides imply flexing of the apoprotein moiety during catalysis.
RecQ helicases: suppressors of tumorigenesis and premature aging
Authors: BACHRATI Csanád Z. , HICKSON Ian D.
Published: September 2003

The RecQ helicases represent a subfamily of DNA helicases that are highly conserved in evolution. Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Germ-line defects in three of the five known human RecQ helicases give rise to defined genetic disorders associated with cancer predisposition and/or premature aging. These are Bloom's syndrome, Werner's syndrome and Rothmund–Thomson syndrome, which are caused by defects in the genes BLM, WRN and RECQ4 respectively. Here we review the properties of RecQ helicases in organisms from bacteria to humans, with an emphasis on the biochemical functions of these enzymes and the range of protein partners that they operate with. We will discuss models in which RecQ helicases are required to protect against replication fork demise, either through prevention of fork breakdown or restoration of productive DNA synthesis.
Spectrin αII and βII isoforms interact with high affinity at the tetramerization site
Authors: BIGNONE Paola A. , BAINES Anthony J.
Published: September 2003

Spectrin tetramers form by the interaction of two α–β dimers through two helices close to the C-terminus of a β subunit and a single helix at the N-terminus of an α subunit. Early work on spectrin from solid tissues (typified by αII and βII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (αI–βI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human α polypeptides (αI and αII) with the C-terminal regions of three β isoforms (βIΣ1, βIIΣ1 and βIIΣ2). αII binds either βII form with a much higher affinity than αI binds βIΣ1 (Kd values of 5–9 nM and 840 nM respectively at 25 °C). βIIΣ1 and βIIΣ2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of α subunit interaction. αII spectrin interacts with each β subunit with higher affinity than αI, and the βII polypeptides have higher affinities for both α chains than βIΣ1. The first full repeat of the α subunit has a major role in determining affinity. Enthalpy changes in the αII–βIIΣ2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of αII–βII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules.
A study of host defence peptide β-defensin 3 in primates
Authors: BONIOTTO Michele , ANTCHEVA Nikolinka , ZELEZETSKY Igor , TOSSI Alessandro , PALUMBO Valeria , VERGA FALZACAPPA Maria Vittoria , SGUBIN Silvia , BRAIDA Laura , AMOROSO Antonio , CROVELLA Sergio
Published: September 2003

We have investigated the molecular evolution of the gene coding for β-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for β-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251–257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17→Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279–8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human β-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.
Ribonuclease III-mediated processing of specific Neisseria meningitidis mRNAs
Authors: DE GREGORIO Eliana , ABRESCIA Chiara , CARLOMAGNO M. Stella , DI NOCERA Pier Paolo
Published: September 2003

Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26–27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis− mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly.
TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP
Authors: BERNARDO M. Margarida , FRIDMAN Rafael
Published: September 2003

The matrix metalloproteinase (MMP)-2 has a crucial role in extracellular matrix degradation associated with cancer metastasis and angiogenesis. The latent form, pro-MMP-2, is activated on the cell surface by the membrane-tethered membrane type 1 (MT1)-MMP, in a process regulated by the tissue inhibitor of metalloproteinase (TIMP)-2. A complex of active MT1-MMP and TIMP-2 binds pro-MMP-2 forming a ternary complex, which permits pro-MMP-2 activation by a TIMP-2-free neighbouring MT1-MMP. It remains unclear how MMP-2 activity in the pericellular space is regulated in the presence of TIMP-2. To address this question, the effect of TIMP-2 on MMP-2 activity in the extracellular space was investigated in live cells, and their isolated plasma membrane fractions, engineered to control the relative levels of MT1-MMP and TIMP-2 expression. We show that both free and inhibited MMP-2 is detected in the medium, and that the net MMP-2 activity correlates with the level of TIMP-2 expression. Studies to displace MT1-MMP-bound TIMP-2 in a purified system with active MMP-2 show minimal displacement of inhibitor, under the experimental conditions, due to the high affinity interaction between TIMP-2 and MT1-MMP. Thus inhibition of MMP-2 activity in the extracellular space is unlikely to result solely as a result of TIMP-2 dissociation from its complex with MT1-MMP. Consistently, immunoblot analyses of plasma membranes, and surface biotinylation experiments show that the level of surface association of TIMP-2 is independent of MT1-MMP expression. Thus low-affinity binding of TIMP-2 to sites distinct to MT1-MMP may have a role in regulating MMP-2 activity in the extracellular space generated by the ternary complex.
Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique
Authors: BOARD Philip G. , TAYLOR Matthew C. , COGGAN Marjorie , PARKER Michael W. , LANTUM Hoffman B. , ANDERS M. W.
Published: September 2003

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.
The metabolism of nitrosothiols in the mycobacteria: identification and characterization of S-nitrosomycothiol reductase
Authors: VOGT Ryan N. , STEENKAMP Daniel J. , ZHENG Renjian , BLANCHARD John S.
Published: September 2003

When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min−1, which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N-hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.
The Dictyostelium class I myosin, MyoD, contains a novel light chain that lacks high-affinity calcium-binding sites
Authors: DE LA ROCHE Marc A. , LEE Sheu-Fen , CÔTÉ Graham P.
Published: September 2003

Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2–4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants (K´d) of 52 μM for Ca2+ and 450 μM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG–MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains.
Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA)
Authors: SARNO Stefania , De MOLINER Erika , RUZZENE Maria , PAGANO Mario A. , BATTISTUTTA Roberto , BAIN Jenny , FABBRO Doriano , SCHOEPFER Joseph , ELLIOTT Matthew , FURET Pascal , MEGGIO Flavio , ZANOTTI Giuseppe , PINNA Lorenzo A.
Published: September 2003

IQA {[5-oxo-5,6-dihydro-indolo(1,2-a)quinazolin-7-yl]acetic acid} is a novel ATP/GTP site-directed inhibitor of CK2 (‘casein kinase 2’), a pleiotropic and constitutively active protein kinase whose activity is abnormally high in transformed cells. The Ki value of IQA (0.17 μM) is lower than those of other CK2 inhibitors reported so far. Tested at 10 μM concentration in the presence of 100 μM ATP, IQA almost suppresses CK2 activity in vitro, whereas it is ineffective or weakly effective on a panel of 44 protein kinases and on phosphoinositide 3-kinase. In comparison, other CK2 inhibitors, notably apigenin and quercetin, are more promiscuous. The in vivo efficacy of IQA has been assessed by using the fact that treatment of Jurkat cells with IQA inhibits endogenous CK2 in a dose-dependent manner. IQA has been co-crystallized with maize CK2α, which is >70% identical with its human homologue, and the structure of the complex has been determined at 1.68 Å (1 Å=0.1 nm) resolution. The inhibitor lies in the same plane occupied by the purine moiety of ATP with its more hydrophobic side facing the hinge region. Major contributions to the interaction are provided by hydrophobic forces and non-polar interactions involving the aromatic portion of the inhibitor and the hydrophobic residues surrounding the ATP-binding pocket, with special reference to the side chains of V53 (Val53), I66, M163 and I174. Consequently, mutants of human CK2α in which either V66 (the homologue of maize CK2α I66) or I174 is replaced by alanine are considerably less sensitive to IQA inhibition when compared with wild-type. These results provide new tools for deciphering the enigmatic role of CK2 in living cells and may pave the way for the development of drugs depending on CK2 activity.