bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Immunodeficiency Virus Type 1 DNA Sequences Genetically Damaged by Hypermutation Are Often Abundant in Patient Peripheral Blood Mononuclear Cells and May Be Generated during Near-Simultaneous Infection and Activation of CD4
+
T Cells
Authors: Janini Mario , Rogers Melissa , Birx Deborah R. , McCutchan Francine E.
Published: July 2002

G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 protease gene amplified from patient PBMC. Among 53 patients, including individuals infected with subtypes A through D and at different clinical stages, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, saturation of G residues in the GA or GG dinucleotide context ranged from 20 to 94%. Levels of other mutants were not elevated, and G-to-A replacement was entirely restricted to GA or GG, and not GC or GT, dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. To investigate the conditions under which hypermutation occurs in cell cultures, purified CD4
+
T cells from normal donors were infected with cloned NL4-3 virus stocks at various times before and after phytohemagglutinin (PHA) activation. Hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 h or more after PHA activation, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC. Near-simultaneous activation and infection of CD4
+
T cells may represent a window of susceptibility where the informational content of HIV-1 sequences is lost due to hypermutation.
Mutation of the Methylated tRNA3Lys Residue A58 Disrupts Reverse Transcription and Inhibits Replication of Human Immunodeficiency Virus Type 1
Authors: Renda Matthew J. , Rosenblatt Joseph D. , Klimatcheva Ekaterina , Demeter Lisa M. , Bambara Robert A. , Planelles Vicente
Published: July 2002

Cellular tRNA

3

Lys

serves as the primer for reverse transcription of human immunodeficiency virus type 1 (HIV-1). tRNA

3

Lys

interacts directly with HIV-1 reverse transcriptase (RT), is packaged into viral particles, and anneals to the primer-binding site (PBS) of the HIV-1 genome in order to initiate reverse transcription. Residue A58 of tRNA

3

Lys

, which lies outside the PBS-complementary region, is posttranscriptionally methylated to form 1-methyladenosine 58 (M
1
A58). This methylation is thought to serve as a pause signal for plus-strand strong-stop DNA synthesis during reverse transcription. However, formal proof that the methylation is necessary for the pausing of RT has not been obtained in vivo. In the present study, we investigated the role of tRNA

3

Lys

residue A58 in the replication cycle of HIV-1 in living cells. We have developed a mutant tRNA

3

Lys

derivative, tRNA

3

Lys

A58U, in which A58 was replaced by U. This mutant tRNA was expressed in CEM cells. We demonstrate that the presence of M
1
A58 is necessary for the appropriate termination of plus-strand strong-stop DNA synthesis and that the absence of M
1
A58 allows RT to read the tRNA sequences beyond residue 58. In addition, we show that replacement of M
1
A58 with U inhibits the replication of HIV-1 in vivo. These results highlight the importance of tRNA primer residue A58 in the reverse transcription process. Inhibition of reverse transcription with mutant tRNA primers constitutes a novel approach for therapeutic intervention against HIV-1.
Role of CD8
+
Lymphocytes in Control of Simian Immunodeficiency Virus Infection and Resistance to Rechallenge after Transient Early Antiretroviral Treatment
Authors: Lifson Jeffrey D. , Rossio Jeffrey L. , Piatak Michael , Parks Thomas , Li Li , Kiser Rebecca , Coalter Vicky , Fisher Brad , Flynn Bernard M. , Czajak Susan , Hirsch Vanessa M. , Reimann Keith A. , Schmitz Joern E. , Ghrayeb John , Bischofberger Norbert , Nowak Martin A. , Desrosiers Ronald C. , Wodarz Dominik
Published: July 2002

Transient antiretroviral treatment with tenofovir, (
R
)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8
+
lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8
+
cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8
+
cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.
The UL6 Gene Product Forms the Portal for Entry of DNA into the Herpes Simplex Virus Capsid
Authors: Newcomb William W. , Juhas Rachel M. , Thomsen Darrell R. , Homa Fred L. , Burch April D. , Weller Sandra K. , Brown Jay C.
Published: July 2002

During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.
Critical Roles of Nuclear Receptor Response Elements in Replication of Hepatitis B Virus
Authors: Yu Xianming , Mertz Janet E.
Published: July 2002

Functional analysis of the roles of the nuclear receptor response elements (NRREs) in the transcription and replication of hepatitis B virus (HBV) in the context of its whole genome has been hampered by the extensive overlapping of the NRREs with the regions encoding viral proteins. We introduced point mutations that inactivate the NRREs individually without altering the open reading frames of viral proteins. These mutations in the context of a plasmid containing 1.2 copies of the HBV genome were transiently transfected into the human hepatoma cell line Huh7. Inactivation of the NRRE in either the preC promoter (NRRE
preC
) or enhancer I (NRRE
enhI
) led to moderate reductions in synthesis of viral RNAs. Concurrent inactivation of both NRREs led to 7- to 8-fold reductions in synthesis of the preC, pregenomic, and preS RNAs and a 15-fold reduction in synthesis of the S RNA. The accumulation of viral DNA in the cytoplasmic nucleocapsids and virion particles in the culture medium was also reduced seven- to eightfold. These results suggest that these NRREs are critical for the efficient propagation of HBV in hepatocytes. In cotransfection experiments we also found that overexpression of PPARα-RXRα in the presence of their respective ligands led to a fourfold increase in pregenomic RNA synthesis and a four- to fivefold increase in viral DNA synthesis, while it had little or no effect on synthesis of the other viral RNAs. Similar effects were observed with overexpression of PPARγ-RXRα in the presence of their respective ligands. This activation was dependent on NRRE
preC
, because the increase in synthesis of viral RNA and DNA was not observed when this site was mutated. Likewise, no activation of synthesis of pregenomic RNA and viral DNA by PPARα-RXRα was observed in a naturally occurring NRRE
preC
−
mutant of HBV. Our results suggest that interactions between nuclear receptors and NRREs present in the HBV genome may play critical roles in regulating its transcription and replication during HBV infection of hepatocytes.
Rabies Virus Ocular Disease: T-Cell-Dependent Protection Is under the Control of Signaling by the p55 Tumor Necrosis Factor Alpha Receptor, p55TNFR
Authors: Camelo Serge , Castellanos Jaime , Lafage Mireille , Lafon Monique
Published: July 2002

Following brain infection, the Challenge Virus Standard strain of rabies virus infects the retina. Rabies virus ocular infection induces the infiltration of neutrophils and predominantly T cells into the eye. The role of tumor necrosis factor alpha (TNF-α)-lymphotoxin signaling in the control of rabies virus ocular infection and inflammatory cell infiltration was assessed using mice lacking the p55 TNF-α receptor (p55TNFR−/−mice). The incidence of ocular disease and the intensity of retinal infection were greater in p55TNFR−/−mice than in C57BL/6 mice: the aggravation correlated with less neutrophil and T-cell infiltration. This indicates that cellular infiltration is under the control of the p55 TNF-α receptor and suggests that inflammatory cells may protect the eye against rabies virus ocular infection. The role of T cells following rabies virus ocular disease was assessed by comparison of rabies virus infection in nude mice with their normal counterparts. Indeed, the incidence and severity of the rabies virus ocular disease were higher in athymic nude mice than in BALB/c mice, indicating that T lymphocytes are protective during rabies virus ocular infection. Moreover, few T cells and neutrophils underwent apoptosis in rabies virus-infected retina. Altogether, these data suggest that T lymphocytes and neutrophils are able to enter the eye, escape the immune privilege status, and limit rabies virus ocular disease. In conclusion, rabies virus-mediated eye disease provides a new model for studying mechanisms regulating immune privilege during viral infection.
Naturally Occurring Deletional Mutation in the C-Terminal Cytoplasmic Tail of CCR5 Affects Surface Trafficking of CCR5
Authors: Shioda Tatsuo , Nakayama Emi E. , Tanaka Yuetsu , Xin Xiaomi , Liu Huanliang , Kawana-Tachikawa Ai , Kato Atsushi , Sakai Yuko , Nagai Yoshiyuki , Iwamoto Aikichi
Published: July 2002

CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(−) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221–222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(−) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4+T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(−) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.
Analyses of Single-Amino-Acid Substitution Mutants of Adenovirus Type 5 E1B-55K Protein
Authors: Shen Yuqiao , Kitzes Galila , Nye Julie A. , Fattaey Ali , Hermiston Terry
Published: July 2002

The E1B-55K protein plays an important role during human adenovirus type 5 productive infection. In the early phase of the viral infection, E1B-55K binds to and inactivates the tumor suppressor protein p53, allowing efficient replication of the virus. During the late phase of infection, E1B-55K is required for efficient nucleocytoplasmic transport and translation of late viral mRNAs, as well as for host cell shutoff. In an effort to separate the p53 binding and inactivation function and the late functions of the E1B-55K protein, we have generated 26 single-amino-acid mutations in the E1B-55K protein. These mutants were characterized for their ability to modulate the p53 level, interact with the E4orf6 protein, mediate viral late-gene expression, and support virus replication in human cancer cells. Of the 26 mutants, 24 can mediate p53 degradation as efficiently as the wild-type protein. Two mutants, R240A (ONYX-051) and H260A (ONYX-053), failed to degrade p53 in the infected cells. In vitro binding assays indicated that R240A and H260A bound p53 poorly compared to the wild-type protein. When interaction with another viral protein, E4orf6, was examined, H260A significantly lost its ability to bind E4orf6, while R240A was fully functional in this interaction. Another mutant, T255A, lost the ability to bind E4orf6, but unexpectedly, viral late-gene expression was not affected. This raised the possibility that the interaction between E1B-55K and E4orf6 was not required for efficient viral mRNA transport. Both R240A and H260A have retained, at least partially, the late functions of wild-type E1B-55K, as determined by the expression of viral late proteins, host cell shutoff, and lack of a cold-sensitive phenotype. Virus expressing R240A (ONYX-051) replicated very efficiently in human cancer cells, while virus expressing H260A (ONYX-053) was attenuated compared to wild-type virus
dl
309 but was more active than ONYX-015. The ability to separate the p53-inactivation activity and the late functions of E1B-55K raises the possibility of generating adenovirus variants that retain the tumor selectivity of ONYX-015 but can replicate more efficiently than ONYX-015 in a broad spectrum of cell types.
The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation
Authors: Tuckis Jeffery , Smallwood Sherin , Feller Joyce A. , Moyer Sue A.
Published: July 2002

The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP
0
binding and abolished the reconstitution of replication from separate P-L and NP
0
-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP
0
-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.
Regulation of Tumor Necrosis Factor Alpha Promoter by Human Parvovirus B19 NS1 through Activation of AP-1 and AP-2
Authors: Fu Yi , Ishii Keiko Kumura , Munakata Yasuhiko , Saitoh Takako , Kaku Mitsuo , Sasaki Takeshi
Published: July 2002

Human parvovirus B19 frequently causes acute and chronic arthritis in adults. The molecular mechanism of B19 arthritis, however, remains poorly understood. We previously showed that the transmission of B19 from rheumatoid synoviocytes to monocytic cells is associated with enhanced secretion of tumor necrosis factor alpha (TNF-α), which triggers inflammation, and interleukin-6. To determine the role of B19 in the production of TNF-α, we focused on the function of its nonstructural protein, NS1, and established monocytic U937 lines transduced with the NS1 gene under the control of an inducible promoter. Production of TNF-α mRNA and protein was elevated in a manner associated with NS1 expression. Reporter assays revealed that AP-1 and AP-2 motifs on the TNF-α promoter were responsible for NS1-mediated up-regulation. Electrophoretic mobility shift assay showed specific binding of nuclear proteins from NS1 gene-transduced cells with the AP-1 or AP-2 probe. Antibodies against transcription factors AP-1 and AP-2 and anti-NS1 antibody inhibited the binding of nuclear proteins to the corresponding probes. These data indicate that NS1 up-regulates TNF-α transcription via activation of AP-1 and AP-2 in monocytic cells. The molecular mechanisms of NS1-mediated TNF-α expression would explain the pathogenesis of B19-associated inflammation.
Scrapie Infectivity in Hamster Blood Is Not Associated with Platelets
Authors: Holada Karel , Vostal Jaroslav G. , Theisen Patrick W. , MacAuley Claudia , Gregori Luisa , Rohwer Robert G.
Published: July 2002

The infectivity of hamster scrapie strain 263K was measured in platelets isolated from blood pooled from six hamsters with clinical scrapie. The total number of infectious doses present in the blood pool was 220, out of which only 3.5 infectious doses were associated with platelets. A larger proportion of the total infectivity was recovered from the mononuclear leukocyte fraction. This result indicates that platelets are not the source of blood-borne infectivity in transmissible spongiform encephalopathy-infected hamsters.
Equilibria of the Reactions Between Nitrogen Oxides and the Sodium Carbonate
Authors: Erdös Emerich , Mocek Karel
Published: July 2002

A general analysis of chemical equilibria in a system consisting of the gaseous nitrogen monoxide, nitrogen dioxide, carbon dioxide, oxygen and of the solid sodium carbonate is carried out with respect to the equilibrium partial pressures of nitrogen oxides. The pertaining equations are derived for three regions of existence of the above system. The results are represented graphically and discussed from the point of view of removing the nitrogen oxides from the waste gases.
Study of Hydrogen Bonding in Carboxylic Acids by the MNDO/M Method
Authors: Bureš Michal , Bezus Jaroslav
Published: July 2002

The semiempirical methods MNDO/M, AM1 and PM3 were applied to the study of hydrogen bonds in carboxylic acids. The calculated hydrogen bond lengths and enthalpies of dimerization were compared with experimental data. The AM1 method fails to properly describe systems with strong hydrogen bonds. The PM3 method predicts the hydrogen bond lengths correctly but underestimates systematically the enthalpies of dimerization. MNDO/M appears to be a promising method for the treatment of association of carboxylic acids.
Metathesis Polymerization of Phenylacetylene by Tungsten Aryloxo Complexes
Authors: Balcar Hynek , Sedláček Jan , Pacovská Marta , Blechta Vratislav
Published: July 2002

Catalytic activity of the tungsten aryloxo complexes WCl5(OAr) and WOCl3(OAr), where Ar = 4-t-C4H9C6H4, 2,6-(t-C4H9)2C6H3, 2,6-Cl2C6H3, 2,4,6-Cl3C6H2, and 2,4,6-Br3C6H2 in polymerization of phenylacetylene (20 °C, monomer to catalyst molar ratio = 1 000) was studied. The activity of WCl5(OAr) as unicomponent catalysts increases with increasing electron withdrawing character of the -OAr ligand. Addition of two equivalents of organotin cocatalysts (Me4Sn, Bu4Sn, Ph4Sn, Bu3SnH) to WCl5(O-C6H2Cl3-2,4 ,6) has only slight positive effect (slightly higher polymer yield and/or molecular weight of poly(phenylacetylene)s was achieved). However, in the case of WOCl3(O-C6H3Cl2-2, 6) catalyst, it enhances the activity considerably by eliminating the induction period. Poly(phenylacetylene)s prepared with the catalysts studied have weight-average molecular weight ranging from 100 000 to 200 000. They are trans-prevailing and have relatively low molar fraction of monomer units comprised in cyclohexadiene sequences (about 6%).
The Interaction of the [B10H10]2- Cage with Lewis Acids and the Formation of Decaborane Derivatives by Cage-Opening Reactions
Authors: Bonnetot Bernard , Miele Philippe , Naoufal Daoud , Mongeot Henri
Published: July 2002

The interaction of hydroborates M2[B10H10] (M = NH4, Et4N, Bu4N, etc.) or Et4N[B10H9-NH2COCH3] with Lewis acids AlX3 (X = Cl, Br, I) in the solid state is sufficiently strong to produce an increase of more than 85 cm-1 of the BH stretching vibrations. A similar interaction was not observed for the hydroborate (NH4)2[B10H9I] owing to the electronegativity of the iodine substituent which hampers the electron donor properties of the cage. Hydrolysis of (AlX3)n-[B10H10]2- adducts under mild conditions results in opening of the boron cage with the formation of halodecaboranes 6-B10H13X. It seems that this reaction proceeds only when the above adducts can form in a preliminary step. Pure hexane solutions of 6-B10H13X were easily obtained with a 45% yield for X = Cl or Br and a 30% yield when X = I.
Synthesis, Spectroscopic Characterization, and Structure of closo-1,10-B10H8F22- and Related Fluorinated Derivatives of B10H102-
Authors: Ivanov Sergei V. , Ivanova Svetlana M. , Miller Susie M. , Anderson Oren P. , Kuznetsov Nikolai T. , Solntsev Konstantin A. , Strauss Steven H.
Published: July 2002

The treatment of salts of the B10H102- anion with the commercially available N-fluoro reagent 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (F-TEDA) resulted in the formation of fluorinated derivatives of this ten-vertex borane dianion. The effects of changing the solvent, temperature, and reagent stoichiometry were studied. The derivatives 1-B10H9F2-, 2-B10H9F2-, 1,2-B10H8F22-, 1,6-B10H8F22-, 1,10-B10H8F22-, and 1,2,10-B10H7F32- were examined by a combination of 11B and 19F NMR spectroscopy and by IR spectroscopy. The salt [Ph4P]2[1,10-B10H8F2] . 3 CH2Cl2 was structurally characterized by single-crystal X-ray crystallography: C51H48B10Cl6F2P2, monoclinic, C2/c, a = 13.8134(9), b = 19.141(1), c = 21.136(1) Å, β = 91.546(1)°, Z = 4, T = -100 °C, R = 0.077.
Multinuclear NMR Studies of Palladium(II) Dihalide Complexes of Dibutyl {α-[4-(Phenyldiazenyl)anilino]benzyl}phosphonate
Authors: Tušek-Božic Ljerka , Curic Manda , Vikic-Topic Dražen , Lyčka Antonín
Published: July 2002

The 1H, 13C, 15N and 31P NMR studies of two types of palladium(II) dihalide complexes of dibutyl {α-[4-(phenyldiazenyl)anilino]benzyl}phosphonate (L) are reported: those with the monodentate trans-bonded ligands through the azo nitrogen, trans-Pd(L)2X2 (X = Cl, Br), and a cyclopalladated binuclear complex in which chloride ions bridge two metal centers, [Pd(L-H)(μ-Cl)]2. The 15N-enriched organophosphorus compound and its palladium(II) complexes were also prepared to enable unambiguous determination of the nitrogen ligation site by 15N NMR. The NMR analysis was accomplished by one- and two-dimensional homo- and heteronuclear experiments including 1H-1H COSY, long-range 1H-1H COSY, NOESY, 1H-13C COSY, long-range 1H-13C COSY, HMQC and HMBC. With the mononuclear complexes, the formation of isomeric species was observed, which are interpreted as rotational isomers caused by restricted rotation around the metal-ligand bond.
High Conformational Stability of Cytochrome P-450 1A2. Evidence from UV Absorption Spectra
Authors: Anzenbacher Pavel , Bec Nicole , Hudeček Jiří , Lange Reinhard , Anzenbacherová Eva
Published: July 2002

The fourth derivative of absorption spectra between 260 and 310 nm were used for monitoring the changes in exposure of tyrosine and tryptophan side chains in cytochrome P-450 1A2 to solvent. Titration of the enzyme with a specific inhibitor, α-naphthoflavone (2-phenylazo[h]chromen-4-one) to inhibitor concentration of 30 μM resulted in small but pronounced changes in derivative spectra (decrease in the maximum amplitude, downshift of the spectral maximum at about 293 nm) corresponding to the exposure of tryptophans towards the solvent. Further addition of the inhibitor led to a decrease of the exposure of these aromatic side-chains. Similar behaviour was also observed in this work for other cytochromes P-450 (2B4 and 11A1). The fourth derivative of absorption spectra was also used to examine the stability of the enzyme both in the presence and absence of α-naphthoflavone, with increasing pressure (up to 400 MPa) and temperature (up to 35 °C) as pertubing factors. The results show that cytochrome P-450 1A2 has a stable conformation as all the conformational changes observed were (spectrally) fully reversible.
Sterically Crowded Heterocycles. XI. A Semiempirical Prediction of Enantiomerization Barriers for Substituted (Z)-3-(Imidazo[1,2-a]pyridin-3-yl)-1-phenylprop-2-en-1-ones
Authors: Böhm Stanislav , Pohl Radek , Kuthan Josef
Published: July 2002

Conformational behaviour and racemization paths of the parent skeleton are discussed using a threedimensional Φ,Ψ-energy map calculated by the semiempirical PM3 method. To restrain the number of possible racemization paths, a less sophisticated approach based on the PM3 heat of formation - torsion angle Φ relationships has been used for 5-substituted title molecules; the role of non-planar enantiomeric transition states is postulated. Plausibility of the simple theoretical procedure has been tested using two phenyl derivatives of the title compounds for which experimental barriers to racemization are accessible.
Extraction of D-Tartaric Acid with Alamine 336/LIX 54 Mixture
Authors: Mishonov Ivo , Procházka Jaroslav , Heyberger Aleš
Published: July 2002

Extraction equilibria in the system aqueous solution of D-tartaric acid-solution of tertiary amine in diluent at 25 °C have been studied. The amine was Alamine 336, a long-chain tertiary amine with C8-C10 alkyls in 2 : 1 ratio; the diluent was chelating extractant LIX 54, a solution of an alkyl β-diketone in kerosene. The experimental results were correlated using a mathematical model which takes into account chemical reactions involved in acid-amine complex formation and non-specific effects of the nonideality of the system.
Benzoylation of Arenes Using Envirocat EPZG® Catalyst and Microwave Irradiation
Authors: Veverková Eva , Gotov Battsengel , Mitterpach Róbert , Toma Štefan
Published: July 2002

Benzoylations of several benzene, naphthalene and thiophene derivatives with benzoyl chloride or benzoic anhydride, catalyzed with EPZG® catalyst and in the absence of solvents were studied under microwave irradiation (MWI). Reasonable to good yields of products were achieved in short times.
Analytical Application of Oligopyrrole Macrocycles
Authors: Záruba Kamil , Setnička Vladimír , Charvátová Jana , Rusin Oleksandr , Tománková Zuzana , Hrdlička Jan , Sýkora David , Král Vladimír
Published: July 2002

Progress of modern analytical chemistry is closely related with advancement in other fields such as organic chemistry and biochemistry. Successful solution of current scientific problems is inconceivable without close cooperation of different chemical disciplines. As an example of such hot and very intricate theme research in the field of molecular recognition of biologically active compounds can serve, where numerous methods of analytical chemistry, organic chemistry and biochemistry can suitably be utilized, elaborated and brought into consonance. This multidisciplinary overlap logically leads to the advent of new scientific fields with their own tools, methodologies and subjects of exploration - bioanalytical chemistry and nanotechnology. This review covers different aspects of analytical application of oligopyrrole macrocycles (mainly porphyrins and sapphyrins). These compounds are widely used in analytical chemistry due to their outstanding optical properties. In our contribution oligopyrrole macrocycles are considered as signaling and structural parts of chemical receptors and selectors in various applications. Introduction of different moieties into meso-position of macrocyclic rings allows to obtain e.g., sterically well-organized receptors for recognition of biologically important analytes, new chromatographic materials, and powerful tools in electrochemical research. Finally, future trends in the field are outlined briefly.
Sport, age, and sex specific incidence of sports injuries in Western Australia
Authors: Stevenson Mark R , Hamer Peter , Finch Caroline F , Elliot Bruce , Kresnow Marcie-jo
Published: July 2002

Objective—To describe the trends in recreational sports injury in Perth, Western Australia.

Design—A prospective cohort study of sports injuries during the 1997 winter season (May to September).

Setting—Sample of Australian football, field hockey, basketball, and netball players from the Perth metropolitan area, Western Australia.

Methods—A cohort of sports participants was followed over the five month winter sports season. Before the season, participants completed a baseline questionnaire and during the season were interviewed every four weeks by telephone.

Results—Overall, 92% of participants (n = 1391) who completed a baseline questionnaire completed at least one follow up telephone interview. About half (51%) of the cohort sustained one or more injuries during the winter season accounting for a total of 1034 injuries. Most injuries were of moderate (58%, n = 598) or minor (40%, n = 412) severity, with only 3% (n = 24) requiring emergency department treatment or a hospital stay. The injury incidence rate was greatest for football (20.3/1000 hours of participation), similar for field hockey and basketball (15.2/1000 hours and 15.1/1000 hours respectively), and lowest for netball (12.1/1000 hours). The incidence of injury was greatest in the first four weeks of the season, and participants aged between 26 and 30 years had about a 55% greater risk of injury than those aged less than 18 years.

Conclusions—This is one of the first studies to show that recreational sports are safe. Although the likelihood of injury was greatest in the first month of the season, few injuries required admission to hospital or emergency department treatment. A greater emphasis on prevention in the early part of the season should help to reduce the elevated incidence of injury found at this time.
Recommendations for HER2 testing in the UK
Authors: Ellis I O , Dowsett M , Bartlett J , Walker R , Cooke T , Gullick W , Gusterson B , Mallon E , Barrett Lee P
Published: July 2002

Determining the HER2 status of breast carcinomas is a prerequisite for the use of the monoclonal antibody trastuzumab (Herceptin®), which has recently been licensed for the treatment of metastatic disease. This necessitates a test based on archival material. The preferred analyses are immunohistochemistry with fluorescent in situ hybridisation (FISH) as a follow up test for ambiguous results. Guidelines have been developed for standardised, well controlled procedures for the provision of reliable results. A group of three reference laboratories has been established to provide advice, quality assurance, and materials, where needed.
Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: comparison of immunohistochemistry assay with fluorescence in situ hybridisation assays
Authors: Wang S , Saboorian M H , Frenkel E , Hynan L , Gokaslan S T , Ashfaq R
Published: July 2002

Aim—To evaluate the clinical usefulness of three commercially available assays for Her-2/neu oncogene and protein measurements. The Her-2/neu protein is overexpressed, mostly as a result of gene amplification, in 20–30% of human breast cancers, and has been shown to have prognostic and predictive value for treatment with chemotherapy or the new monoclonal antibody, Herceptin.

Methods—An immunohistochemistry (IHC) assay using the Dako polyclonal antibody A0485, which measures the Her-2/neu protein, was compared with two new Food and Drug Administration (FDA) approved fluorescence in situ hybridisation (FISH) assays—INFORM™ and PathVysion™, in a cohort of 52 formalin fixed, paraffin wax embedded breast tissues. These tissues were selected randomly from 84 consecutive infiltrating breast cancer specimens, which were first stratified according to the Her-2/neu protein levels as measured by IHC.

Results—The two FISH assays achieved a 98% concordance rate: 14 specimens (27%) showed Her-2/neu gene amplification and 37 specimens (71%) showed no Her-2/neu gene amplification. The PathVysion assay had certain advantages over the INFORM assay. In contrast, the IHC assay detected Her-2/neu overexpression in a high percentage of cases, including 13 high positive specimens (25%) and 13 medium positive specimens (25%). Although 10 of these 13 IHC high positive specimens showed gene amplification by FISH, nine of 13 IHC medium positive specimens showed no gene amplification. Statistical analyses showed that the differences between IHC and FISH assays were primarily in the specimens with medium positive IHC, but negative FISH results.

Conclusions—Because of the increasing importance of the Her-2/neu oncogene and oncoprotein in the clinical management of patients with breast cancer, the accurate and consistent evaluation of Her-2/neu status is crucial. This study suggests that the best approach is to combine both IHC and FISH assays; that is, to use the IHC assay as a triage step, followed by the PathVysion FISH assay to analyse the IHC medium and high positive cases.
A protocol for consultation of another physician in cases of euthanasia and assisted suicide
Authors: Onwuteaka-Philipsen Bregje D , van der Wal Gerrit
Published: July 2002

Objective—Consultation of another physician is an important method of review of the practice of euthanasia. For the project “support and consultation in euthanasia in Amsterdam” which is aimed at professionalising consultation, a protocol for consultation was developed to support the general practitioners who were going to work as consultants and to ensure uniformity.

Participants—Ten experts (including general practitioners who were experienced in euthanasia and consultation, a psychiatrist, a social geriatrician, a professor in health law and a public prosecutor) and the general practitioners who were going to use the protocol.

Evidence—There is limited literature on consultation: discursive articles and empirical studies describing the practice of euthanasia.

Consensus—An initial draft on the basis of the literature was commented on by the experts and general practitioners in two rounds. Finally, the protocol was amended after it had been used during the training of consultants.

Conclusions—The protocol differentiates between steps that are necessary in a consultation and steps that are recommended. Guidelines about four important aspects of consultation were given: independence, expertise, tasks and judgment of the consultant. In 97% of 109 consultations in which the protocol was used the consultant considered the protocol to be useful to a greater or lesser extent. Although this protocol was developed locally, it also employs universal principles. Therefore it can be of use in the development of consultation elsewhere.
Molecular genetic heterogeneity in autosomal dominant drusen
Authors: Tarttelin Emma E , Gregory-Evans Cheryl Y , Bird Alan C , Weleber Richard G , Klein Michael L , Blackburn James , Gregory-Evans Kevin
Published: July 2002

OBJECTIVE

Autosomal dominant drusen is of particular interest because of its phenotypic similarity to age related macular degeneration. Currently, mutation R345W ofEFEMP1 and, in a single pedigree, linkage to chromosome 6q14 have been causally related to the disease. We proposed to investigate and quantify the roles ofEFEMP1 and the 6q14 locus in dominant drusen patients from the UK and USA.

DESIGN

Molecular genetic analysis.

PARTICIPANTS

Ten unrelated families and 17 young drusen patients.

MAIN OUTCOME MEASURES

Exons 1 and 2 of EFEMP1 were characterised by 5′ rapid amplification of cDNA ends and direct sequencing. Exons 1-12 ofEFEMP1 were then investigated for mutation by direct sequencing. A HpaII restriction digest test was constructed to detect the EFEMP1R345W mutation. Marker loci spanning the two dominant drusen linked loci were used to generate haplotype data.

RESULTS

Only seven of the 10 families (70%) and one of the 17 sporadic patients (6%) had the R345W mutation. The HpaII restriction digest test was found to be a reliable and quick method for detecting this. No other exonic or splice site mutation was identified. Of the three families without EFEMP1 mutation, two were linked to the 2p16 region.

CONCLUSIONS

EFEMP1R345W accounts for only a proportion of the dominant drusen phenotype. Importantly, other families linked to chromosome 2p16 raise the possibility of EFEMP1 promoter sequence mutation or a second dominant drusen gene at this locus. Preliminary haplotype data suggest that the disease gene at the 6q14 locus is responsible for only a minority of dominant drusen cases.
Neurobehavioural effects of occupational exposure to cadmium: a cross sectional epidemiological study
Authors: Viaene M K , Masschelein R , Leenders J , De Groof M , Swerts L J V C , Roels H A
Published: July 2002

BACKGROUND

A patient with unexplained minor behavioural changes associated with an axonal sensorimotor polyneuropathy had a history of chronic occupational exposure to cadmium (Cd). Although animal studies have shown that Cd is a potent neurotoxicant, little is known about its toxicity for the human central nervous system. The aim of this study was to investigate the toxic potential of chronic occupational exposure to Cd on neurobehavioural functions.

METHODS

A cross sectional epidemiological study was conducted in a group of Cd workers and an age matched control group. Eighty nine adult men (42 exposed to Cd and 47 control workers) were given a blinded standardised examination that consisted of computer assisted neurobehavioural tests (neurobehavioural examination system), a validated questionnaire to assess neurotoxic complaints (neurotoxicity symptom checklist-60, NSC-60), and a standardised self administered questionnaire to detect complaints consistent with peripheral neuropathy and dysfunction of the autonomic nervous system. Historical and current data on biomonitoring of exposure to Cd, either the highest value of Cd in urine (CdU in μg Cd/g creatinine) of each Cd worker during work (CdUmax) or the current value (CdUcurrent) of each control, were available as well as data on microproteinuria.

RESULTS

Cd workers (CdUmax: mean (range), 12.6 (0.4–38.4)) performed worse than the controls (CdUcurrent: mean (range), 0.7 (0.1–2.0)) on visuomotor tasks, symbol digit substitution (p=0.008), and simple reaction time to direction (p=0.058) or location (p=0.042) of a stimulus. In multiple linear regression analysis, symbol digit substitution, simple direction reaction time test, and simple location reaction time test were significantly related to CdUmax, (β=0.35 ( p<0.001), β= 0.25 (p=0.012), and β=0.23 (p=0.021) respectively). More complaints consistent with peripheral neuropathy (p=0.004), complaints about equilibrium (p=0.015), and complaints about concentration ability (p=0.053) were found in the group exposed to Cd than in the control group, and these variables correlated positively with CdUmax(peripheral neuropathy: β=0.38, p<0.001; equilibrium: β=0.22, p=0.057; concentration ability: β=0.27, p=0.020).

CONCLUSION

Slowing of visuomotor functioning on neurobehavioural testing and increase in complaints consistent with peripheral neuropathy, complaints about equilibrium, and complaints about concentration ability were dose dependently associated with CdU. Age, exposure to other neurotoxicants, or status of renal function could not explain these findings. The present study also indicates that an excess of complaints may be detected in Cd workers before signs of microproteinuria induced by Cd occur.
Dermal in vitro penetration of methiocarb, paclobutrazol, and pirimicarb
Authors: Nielsen Jesper B , Nielsen Flemming
Published: July 2002

OBJECTIVES

The dominant route of occupational exposure to pesticides in horticulture is dermal. However, preventive measures are seldom used when handling plant cultures recently treated with pesticides, thus causing significant dermal exposure and potential absorption. Assessment of exposure often depends on biological monitoring of blood or urine samples. The skin often acts as a temporary reservoir for chemicals before absorption. Failure to consider the lag time between dermal exposure and appearance of pesticide or metabolites in the general circulation may lead to false conclusions about assessment of exposure.

METHODS

In an experimental model in which in vitro static diffusion cells were mounted with human skin, dermal penetration of three extensively used pesticides (methiocarb, paclobutrazol, pirimicarb) was evaluated.

RESULTS

Pirimicarb and paclobutrazol had comparable rates of dermal penetration and lag times of around 18 hours. Methiocarb had a considerably shorter lag time. Dermal penetration continued for extended periods after exposure had ended.

CONCLUSIONS

With lag times sometimes considerably longer than a normal working day, biological monitoring at the end of exposure may seriously underestimate the actual exposure. There may be implications for regulatory guidelines, which often require only 24 hour observation periods.
Spontaneous necrosis of parathyroid adenoma: biochemical and imaging follow-up for two years
Authors: Chan W B , Chow C C , King A D , Yeung V T F , Li J K Y , So W Y , Cockram C S
Published: July 2002

We report a patient with a biochemically and radiologically confirmed parathyroid adenoma, which underwent spontaneous resolution by necrosis. The patient was followed-up over the subsequent two years during which time the tumour and hypercalcaemia recurred. Sequential radiological and biochemical changes at the time of diagnosis, spontaneous necrosis and recurrence are documented fully.
Doctor or nurse? The patients' choice
Authors: Read A M , Mayberry J F
Published: July 2002

This project's aim was to assess patients perceived need for a specialist nurse in inflammatory bowel disease. A letter was sent to 64 patients with a summary of the potential role of a specialist nurse. Patients were asked to complete a questionnaire of 10 scenarios on who would be their preferred provider on the range of issues. There were 35 (55%) replies. When comparing the results between the consultant and the specialist nurse patients preferred to see a specialist nurse significantly in four scenarios, and patients preferred to see the consultant significantly in two scenarios. The results indicate that patients feel specialist nurses would have more time to discuss issues and they do not want to bother the busy doctors with them. But they do want doctors to carry out the medical aspects of their care.
Heterogeneity of anti-PR3 associated disease in Hong Kong
Authors: Lee Shui-shan , Lawton John W M
Published: July 2002

Thirty seven patients tested positive for antibody against proteinase 3 (anti-PR3) using either one of the two in-house ELISA methods at the Immunology Laboratory, University of Hong Kong. All except three were Chinese. Systemic vasculitis was diagnosed in 13 patients. However, the positive predictive value of anti-PR3 for Wegener's granulomatosus was low (22%). The commonest presenting feature was respiratory (32%). Both respiratory and renal symptoms were commonly reported in anti-PR3 positive patients as in previous studies. In this largely Chinese population, there is marked variability in the spectrum of diseases associated with anti-PR3; though many had vasculitic diseases, some were diagnosed with other conditions such as inflammatory bowel diseases and respiratory infection. In contrast with series in white people, antimyeloperoxidase rather than anti-PR3 associated disease is more prevalent in Chinese patients.