bluuebunny/crossref_metadata_2025 huggingface dataset:
Oversynthesis of a New
Escherichia coli
Small RNA Suppresses Export Toxicity of DsbA′-PhoA Unfoldable Periplasmic Proteins
Authors: Guigueno Agnès , Dassa Janie , Belin Pascal , Boquet Paul Louis
Published: July 2002

In
Escherichia coli
, the DsbA′-PhoA hybrid proteins carrying an unfoldable DsbA′ fragment can be targeted to the envelope, where they exert their toxicity. Hybrid proteins stick to the periplasmic face of the inner membrane and paralyze the export mechanism, becoming lethal if sufficiently overproduced and if not degraded by the DegP protease (A. Guigueno, P. Belin, and P. L. Boquet, J. Bacteriol. 179:3260–3269, 1997). We isolated a multicopy suppressor that restores viability to a
degP
strain without modifying the expression level of the toxic fusion. Suppression does not involve activation of the known envelope stress-combative pathways, the Cpx pathway and the ς
E
regulon. Subclone analysis of the suppressor revealed a 195-bp DNA fragment that is responsible for toxicity suppression. The cloned gene, called
uptR
, is ≈130 bp long (including the promoter and a transcription termination signal) and is transcribed into a small RNA (92 nucleotides). Using site-directed mutagenesis, we found that UptR RNA does not require translation for toxicity suppression. UptR-mediated action reduces the amount of membrane-bound toxic hybrid protein. UptR RNA is the first example of a small RNA implicated in extracytoplasmic toxicity suppression. It appears to offer a new way of suppressing toxicity, and its possible modes of action are discussed.
Altered Pathway Routing in a Class of
Salmonella enterica
Serovar Typhimurium Mutants Defective in Aminoimidazole Ribonucleotide Synthetase
Authors: Zilles Julie L. , Kappock T. Joseph , Stubbe JoAnne , Downs Diana M.
Published: July 2002

In
Salmonella enterica
serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the
purI
gene in
S. enterica
. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways,
purI
mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37–44, 1996). Unexpectedly, some mutant
purI
alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The

K
m

of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the
V
max
was decreased 2.5-fold. The

K
m

for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and
purR
null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.
Growth Inhibition Caused by Overexpression of the Structural Gene for Glutamate Dehydrogenase (
gdhA
) from
Klebsiella aerogenes
Authors: Janes Brian K. , Pomposiello Pablo J. , Perez-Matos Ana , Najarian David J. , Goss Thomas J. , Bender Robert A.
Published: July 2002

Two linked mutations affecting glutamate dehydrogenase (GDH) formation (
gdh-1
and
rev-2
) had been isolated at a locus near the
trp
cluster in
Klebsiella aerogenes
. The properties of these two mutations were consistent with those of a locus containing either a regulatory gene or a structural gene. The
gdhA
gene from
K. aerogenes
was cloned and sequenced, and an insertion mutation was generated and shown to be linked to
trp
. A region of
gdhA
from a strain bearing
gdh-1
was sequenced and shown to have a single-base-pair change, confirming that the locus defined by
gdh-1
is the structural gene for GDH. Mutants with the same phenotype as
rev-2
were isolated, and their sequences showed that the mutations were located in the promoter region of the
gdhA
gene. The linkage of
gdhA
to
trp
in
K. aerogenes
was explained by postulating an inversion of the genetic map relative to other enteric bacteria. Strains that bore high-copy-number clones of
gdhA
displayed an auxotrophy that was interpreted as a limitation for α-ketoglutarate and consequently for succinyl-coenzyme A (CoA). Three lines of evidence supported this interpretation: high-copy-number clones of the enzymatically inactive
gdhA1
allele showed no auxotrophy, repression of GDH expression by the nitrogen assimilation control protein (NAC) relieved the auxotrophy, and addition of compounds that could increase the α-ketoglutarate supply or reduce the succinyl-CoA requirement relieved the auxotrophy.
Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from
Pyrococcus furiosus
Authors: Verhees Corné H. , Akerboom Jasper , Schiltz Emile , de Vos Willem M. , van der Oost John
Published: July 2002

The
Pyrococcus furiosus fbpA
gene was cloned and expressed in
Escherichia coli
, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a

K
m

of 0.32 mM and a
V
max
of 12.2 U/mg. The
P. furiosus
fructose-1,6-bisphosphatase was strongly inhibited by Li
+
(50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the
P. furiosus
fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.
Identification and Characterization of Assembly Proteins of CS5 Pili from Enterotoxigenic
Escherichia coli
Authors: Duthy Thomas G. , Manning Paul A. , Heuzenroeder Michael W.
Published: July 2002

This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic
Escherichia coli
. In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in
csfB
abolished CsfA major subunit accumulation in the periplasm, which could be restored by
trans
-complementation with a complete copy of the
csfB
gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the
csfE
gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of
csfE
expression in
trans
did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the
csfF
gene also resulted in an elongated pilus morphology identical to that of the
csfE
deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a
csfF
deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the
csfB
and
csfF
genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was
trans
-complemented with a wild-type copy of
csfB
or
csfF
, respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations.
A Turquoise Mutant Genetically Separates Expression of Genes Encoding Phycoerythrin and Its Associated Linker Peptides
Authors: Seib Laura Ort , Kehoe David M.
Published: July 2002

During complementary chromatic adaptation (CCA), cyanobacterial light harvesting structures called phycobilisomes are restructured in response to ambient light quality shifts. Transcription of genes encoding components of the phycobilisome is differentially regulated during this process: red light activates
cpcB2A2
, whereas green light coordinately activates the
cpeCDE
and
cpeBA
operons. Three signal transduction components that regulate CCA have been isolated to date: a sensor-photoreceptor (RcaE) and two response regulators (RcaF and RcaC). Mutations in the genes encoding these components affect the accumulation of both
cpcB2A2
and
cpeBA
gene products. We have isolated and characterized a new pigmentation mutant called Turquoise 1. We demonstrate that this mutant phenotype is due to a dramatic decrease in
cpeBA
transcript abundance and results from a lesion in the
cpeR
gene. However, in this mutant
cpeCDE
RNA levels remain near those found in wild-type cells. Our results show that the coordinate regulation of
cpeBA
and
cpeCDE
by green light can be uncoupled by the loss of CpeR, and we furnish the first genetic evidence that different regulatory mechanisms control these two operons. Sequence analysis of CpeR reveals that it shares limited sequence similarity to members of the PP2C class of protein serine/threonine phosphatases. We also demonstrate that
cpeBA
and
cpeCDE
retain light quality responsiveness in a mutant lacking the RcaE photoreceptor. This provides compelling evidence for the partial control of CCA through an as-yet-uncharacterized second light quality sensing system.
Effect of
aniA
(Carbon Flux Regulator) and
phaC
(Poly-β-Hydroxybutyrate Synthase) Mutations on Pyruvate Metabolism in
Rhizobium etli
Authors: Dunn Michael F. , Araíza Gisela , Encarnación Sergio , del Carmen Vargas María , Mora Jaime
Published: July 2002

The
Rhizobium etli
poly-β-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of
aniA
, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity,
pyc
gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA,
pyc
transcription, and reduced-nucleotide levels is discussed.
Isolation of an Intron-Containing Partial Sequence of the Gene Encoding Dermatophyte Actin (
ACT
) and Detection of a Fragment of the Transcript by Reverse Transcription-Nested PCR as a Means of Assessing the Viability of Dermatophytes in Skin Scales
Authors: Okeke Charles N. , Tsuboi Ryoji , Kawai Masaaki , Hiruma Masataro , Ogawa Hideoki
Published: July 2002

An internal partial sequence of the gene encoding actin (
ACT
), 725 to 762 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced. An intron that is 56 to 93 bp in length was located along the
ACT
fragment of all of the dermatophytes at codon position 301 (−3) (a codon number followed by “−3” indicates that the intron directly follows the codon) with reference to the amino acid sequence of human α-smooth muscle actin. A primer pair that annealed to exon sequences flanking the
ACT
-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-PCR) of dermatophyte
ACT
mRNA. PCR primer pairs with antisense sequence based on the
ACT
intron sequence were species specific for dermatophytes, suggesting a potential for use in the identification of dermatophytes. The viability of dermatophytes in skin scales was subsequently assessed by the presence of
ACT
mRNA in total RNA extracted from a 48-h culture of scale samples in 250 μl of yeast carbon base broth. RT-nested PCR of dermatophyte-infected samples amplified an
ACT
fragment of the predicted size of 171 bp. The results of viability testing based on
ACT
mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics.
Widespread Occurrence of
Pneumocystis carinii
in Commercial Rat Colonies Detected Using Targeted PCR and Oral Swabs
Authors: Icenhour Crystal R. , Rebholz Sandra L. , Collins Margaret S. , Cushion Melanie T.
Published: July 2002

The genus
Pneumocystis
contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with
Pneumocystis
becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of
Pneumocystis
in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of
Pneumocystis
in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke
Pneumocystis
growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of
Pneumocystis
by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of
Pneumocystis carinii
f. sp.
carinii
. Upon receipt, 64% of rats were positive for
P. carinii
f. sp.
carinii
-specific antibodies, while
P. carinii
f. sp.
carinii
DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed
P. carinii
f. sp.
carinii
DNA in all rat lungs, illustrating widespread occurrence of
Pneumocystis
in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of
Pneumocystis
exposure.
Pretreatment with Urea-Hydrochloric Acid Enhances the Isolation of
Helicobacter pylori
from Contaminated Specimens
Authors: Song Qunsheng , Zirnstein Gerald W. , Swaminathan Bala , Gold Benjamin D.
Published: July 2002

Human saliva seeded with
H. pylori
was incubated in urea-HCl and then cultured on nonselective media. Pretreatment with 0.06 N HCl–0.08 M urea for 5 min at 37°C resulted in reproducible isolation of
H. pylori
, even at low inocula (≤10
2
CFU/ml of saliva), despite the presence of large numbers of contaminating organisms.
Development of Conventional and Real-Time PCR Assays for Detection of
Legionella
DNA in Respiratory Specimens
Authors: Rantakokko-Jalava Kaisu , Jalava Jari
Published: July 2002

The development and validation of a PCR assay based on the use of new 16S ribosomal DNA (rDNA)-targeted primers to detect
Legionella
DNA in respiratory specimens are described. The assay was originally developed as conventional PCR followed by electrophoretic detection and was then adapted to Lightcycler format with SYBR Green I detection and melting curve analysis. The 73
Legionella pneumophila
strains tested were amplified with both applications. In addition, 21 and 23 out of 27 other
Legionella
strains were found positive by conventional and real-time PCR assays, respectively, including the clinically important species
L. micdadei,
L. bozemaniae
,
and
L. dumoffii
. Two DNA purification methods were compared using artificially seeded clinical specimens: a standard organic extraction method and a commercial kit based on adsorption of DNA to silica particles. The detection limit of the assay varied from 2 CFU to >200,000 CFU per ml of clinical specimen, depending on the background sample (i.e., pooled sputa or BAL fluids) and the DNA purification method, the silica method achieving lower detection limits. Analysis of 77 clinical samples (66 bronchoalveolar lavage fluid and 11 sputum samples) by conventional PCR yielded results that were consistent with
Legionella
culture results. The melting curve analysis in the Lightcycler system readily detected the specific amplification products. However, run-to-run variations in the measured melting temperatures required normalization against the standard sample in each run. The results obtained with the clinical specimens were similar to those obtained with conventional PCR, but more samples are required to determine whether the system can be applied to routine screening of samples for the presence of
Legionella
DNA.
Existence of Distinct GB Virus C/Hepatitis G Virus Variants with Different Tropism
Authors: Fogeda Marta , López-Alcorocho Juan Manuel , Bartolomé Javier , Arocena Carlos , Martín Maria Angeles , Carreño Vicente
Published: July 2002

To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV) variants with different tropism, we have analyzed the heterogeneity and quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and PBMC from two chronically infected patients. For this purpose, the GBV-C/HGV 5′ noncoding region (5′NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5′NCR sequences isolated from the in vitro-infected PBMC and from the PBMC isolated from the patient whose serum was used as the inoculum were identical but different from that of the inoculum. Furthermore, phylogenetic analysis revealed that all PBMC sequences grouped together into a branch which was separate from those of the inoculum. For one of the two chronically infected patients, all the sequences from the PBMC and one from the liver clustered into a single branch while the sequences from the serum and all the other liver sequences grouped together in the other branch. For the other patient, the sequences from the serum and PBMC and three sequences from the liver grouped together into one branch, while the remaining five sequences from the liver were separated in a different cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.
Sequences in the 5′ and 3′ R Elements of Human Immunodeficiency Virus Type 1 Critical for Efficient Reverse Transcription
Authors: Ohi Yuki , Clever Jared L.
Published: July 2002

The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (−sssDNA) is transferred from the 5′ end to the 3′ end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5′, the 3′, or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5′ or the 3′ R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length −sssDNA and 3′ acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5′ or the 3′ R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5′, the 3′, or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the −sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5′ R.
LMP1 of Epstein-Barr Virus Induces Proliferation of Primary Mouse Embryonic Fibroblasts and Cooperatively Transforms the Cells with a p16-Insensitive CDK4 Oncogene
Authors: Yang Xinhai , Sham Jonathan S. T. , Ng M. H. , Tsao Sai-Wah , Zhang Dekai , Lowe Scott W. , Cao Liang
Published: July 2002

The latent membrane protein LMP1 of Epstein-Barr virus (EBV) is often present in EBV-associated malignancies including nasopharyngeal carcinoma and Hodgkin's lymphoma. Previous work demonstrates that the LMP1 gene of EBV is sufficient to transform certain established rodent fibroblast cell lines and to induce the tumorigenicity of some human epithelial cell lines. In addition, LMP1 plays pleiotropic roles in cell growth arrest, differentiation, and apoptosis, depending on the background of the target cells. To examine the roles of LMP1 in cell proliferation and growth regulation in primary culture cells, we constructed a recombinant retrovirus containing an LMP1 gene. With this retrovirus, LMP1 was shown to stimulate the proliferation of primary mouse embryonic fibroblasts (MEF cells). It has a mitogenic activity for MEF cells, as demonstrated by an immediate induction of cell doubling time. In addition, it significantly extends the passage number of MEF cells to more than 30 after retroviral infection, compared with less than 5 for uninfected MEF cells. Furthermore, LMP1 cooperates with a p16-insensitive CDK4

R24C

oncogene in transforming MEF cells. Our results provide the first evidence of the abilities of the LMP1 gene, acting alone, to effectively induce the proliferation of primary MEF cells and of its cooperativity with another cellular oncogene in transforming primary cells.
Mutations in the PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduce Virus Budding by Inhibiting a Late Step in Virion Release
Authors: Jayakar Himangi R. , Murti K. Gopal , Whitt Michael A.
Published: July 2002

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.
Host Sequences Flanking the Human T-Cell Leukemia Virus Type 1 Provirus In Vivo
Authors: Leclercq India , Mortreux Franck , Cavrois Marielle , Leroy Arnaud , Gessain Antoine , Wain-Hobson Simon , Wattel Eric
Published: July 2002

Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from ≥200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.
Efficient Lymphoreticular Prion Propagation Requires PrPcin Stromal and Hematopoietic Cells
Authors: Kaeser Pascal S. , Klein Michael A. , Schwarz Petra , Aguzzi Adriano
Published: July 2002

In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrPc). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrPc(Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrPcon hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation ofPrnpin either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrPcexpression by both stromal and hematopoietic compartments.
Analysis of Virus-Specific CD4+T Cells during Long-Term Gammaherpesvirus Infection
Authors: Flaño Emilio , Woodland David L. , Blackman Marcia A. , Doherty Peter C.
Published: July 2002

Major histocompatibility complex class II-mediated antigen presentation after intranasal infection with murine gammaherpesvirus 68 differs in mediastinal lymph nodes and spleen. Evidence that virus-specific CD4+T cells were being stimulated was found as late as 6 to 8 months after infection, and cells specific for the viral gp15067–83and ORF11168–180peptides were maintained as a fairly stable proportion of the total response.
Detection of Diverse Hepatitis C Virus (HCV)-Specific Cytotoxic T Lymphocytes in Peripheral Blood of Infected Persons by Screening for Responses to All Translated Proteins of HCV
Authors: Wong David K. H. , Dudley Darryll D. , Dohrenwend Paul B. , Lauer Georg M. , Chung Raymond T. , Thomas David L. , Walker Bruce D.
Published: July 2002

Broadly directed hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTL) have been identified from liver-infiltrating lymphocytes but have been more difficult to assess in peripheral blood of infected persons. To enhance the detection of CTL from peripheral blood mononuclear cells (PBMC), we cocultured PBMC with autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines that had been infected with recombinant vaccinia virus constructs so that they expressed the entire translated polyprotein of HCV-H, a type 1a strain. These stimulated cells from HCV-infected as well as exposed seronegative persons were then cloned at limiting dilution and tested for HCV-specific CTL activity using a standard51Cr release assay. HCV-specific CTL were detected in PBMC from seven of nine persons with chronic hepatitis, including five of seven in whom CTL had previously been detected from liver biopsy specimens but not PBMC. In a single person with chronic HCV infection, CTL directed against as many as five different epitopes were detected in peripheral blood and were similar in specificity to those detected in liver tissue. This technique was used to evaluate eight subjects identified to be at high risk for HCV exposure due to continued injection drug abuse; no evidence of CTL in PBMC was found. We conclude that CTL can be detected in PBMC from the majority of persons with chronic HCV infection but are present at lower levels or absent in exposed but persistently seronegative persons. The high degree of concordance of HCV epitopes identified from liver and PBMC suggests that this strategy is a reasonable alternative to liver biopsy for characterizing the CTL response to HCV in chronically infected persons.
Identification and Antigenicity of Broadly Cross-Reactive and Conserved Human Immunodeficiency Virus Type 1-Derived Helper T-Lymphocyte Epitopes
Authors: Wilson Cara C. , Palmer Brent , Southwood Scott , Sidney John , Higashimoto Yuichiro , Appella Ettore , Chesnut Robert , Sette Alessandro , Livingston Brian D.
Published: July 2002

Human immunodeficiency virus (HIV)-specific helper T lymphocytes (HTL) play a key role in the immune control of HIV type 1 (HIV-1) infection, and as such are an important target of potential HIV-1 vaccines. In order to identify HTL epitopes in HIV-1 that might serve as vaccine targets, conserved HIV-1-derived peptides bearing an HLA-DR binding supermotif were tested for binding to a panel of the most representative HLA-DR molecules. Eleven highly cross-reactive binding peptides were identified: three in Gag and eight in Pol. Lymphoproliferative responses to this panel of peptides, as well as to the HIV-1 p24 and p66 proteins, were evaluated with a cohort of 31 HIV-1-infected patients. All 11 peptides were recognized by peripheral blood mononuclear cells from multiple HIV-infected donors. Many of the responsive HIV-infected subjects showed recognition of multiple peptides, indicating that HIV-1-specific T-helper responses may be broadly directed in certain individuals. A strong association existed between recognition of the parental recombinant HIV-1 protein and the corresponding HTL peptides, suggesting that these peptides represent epitopes that are processed and presented during the course of HIV-1 infection. Lastly, responses to the supermotif peptides were mediated by CD4+T cells and were restricted by major histocompatibility complex class II molecules. The epitopes described herein are potentially important components of HIV-1 therapeutic and prophylactic vaccines.
Regulation of Human Immunodeficiency Virus Type 1 Infection, β-Chemokine Production, and CCR5 Expression in CD40L-Stimulated Macrophages: Immune Control of Viral Entry
Authors: Cotter Robin L. , Zheng Jialin , Che Myhanh , Niemann Douglas , Liu Ying , He Johnny , Thomas Elaine , Gendelman Howard E.
Published: July 2002

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and β-chemokine production, and β-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of β-chemokines (macrophage inhibitory proteins 1α and 1β and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-α led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the β-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.
Permanganate Oxidation of L-Alanine in Acidic Solvent: Autocatalysis by Intermediate Mn(IV) Species
Authors: Insausti Maria J. , Mata-Pérez Fernando , Alvarez-Macho Maria P.
Published: July 2002

The kinetics of L-alanine oxidation by permanganate ion in buffer solutions at pH 2.2 have been studied. Both spectrophotometric and kinetic evidence is provided for the existence of an intermediate Mn(IV) species of colloidal nature. An autocatalytic course of the oxidation is discussed.
Synthesis of Deoxy, Dideoxy and Didehydrodideoxy Analogs of 9-(β-D-Hexofuranosyl)adenine
Authors: Hřebabecký Hubert , Dočkal Jan , Holý Antonín
Published: July 2002

Condensation of 1,2-di-O-acetyl-3,5,6-tri-O-benzoyl-D-glucofuranose with N6-benzoyladenine, catalyzed with tin tetrachloride, afforded nucleoside I. Partial deacetylation of I, followed by mesylation, gave 9-(3,5,6-tri-O-benzoyl-2-O-methanesulfonyl-β-D-glucofuranosyl)adenine (III). 9-(2,5,6-Tri-O-acetyl-3-O-methanesulfonyl-β-D-glucofuranosyl)-N6-benzoyladenine (IV) was prepared by condensation of 1,2,5,6-tetra-O-acetyl-3-O-methanesulfonyl-D-glucofuranose with N6-benzoyladenine. Reaction of mesyl derivative III with methanolic sodium methoxide and of mesyl derivative IV with methanolic ammonia led to 2',3'-anhydronucleosides V and VI which were acetylated to give the respective 9-(5,6-di-O-acetyl-2,3-anhydro-β-D-mannofuranosyl)adenine (VII) and 9-(5,6-di-O-acetyl-2,3-anhydro-β-D-allofuranosyl)adenine (VIII). Epoxy derivative VII was cleaved with bromotrimethylsilane, affording a mixture of 9-(5,6-di-O-acetyl-2-bromo-2-deoxy-β-D-glucofuranosyl)adenine (Xa) and 9-(5,6-di-O-acetyl-3-bromo-3-deoxy-β-D-altrofuranosyl)adenine (XIa), epoxy derivative VIII was cleaved analogously to give 9-(5,6-di-O-acetyl-3-bromo-3-deoxy-β-D-glucofuranosyl)adenine (XIIa). Their dehalogenation with tributylstannane and subsequent deacetylation led to 9-(2-deoxy-β-D-arabino-hexofuranosyl)adenine (Xc), 9-(3-deoxy-β-D-arabino-hexofuranosyl)adenine (XIc) and 9-(3-deoxy-β-D-ribo-hexofuranosyl)adenine (XIIc). 9-(2,5,6-Tri-O-acetyl-3-bromo-3-deoxy-β-D-glucofuranosyl)adenine (XIId), which was prepared by acetylation of XIIa, on reductive elimination with Cu/Zn couple and subsequent deacetylation gave 9-(2,3-dideoxy-β-D-erythro-hex-2-enofuranosyl)adenine (XIV). 9-(2,3-Dideoxy-β-D-erythro-hexofuranosyl)adenine (XVI) was obtained either by catalytic hydrogenation of bromo derivative XIId followed by deacetylation, or by catalytic hydrogenation of didehydro derivative XIV. The synthesized nucleosides were tested for antiviral activity.
Synthesis of Bicyclic Pyridine Tripeptides
Authors: Attia Abd El-Hamid , Abo-Ghalia Mohamed H. , Abd El-Salam Osama I.
Published: July 2002

Dinicotinic acid reacted with L-phenylalanine affording N-dinicotinoyl-bis-L-phenylalanine (VII). The same product was also obtained by mild alkaline hydrolysis of the corresponding ester VI. Coupling of VII with L-ornithine or L-ornithine methyl ester gave rise to the formation of the desired chiral bicyclic tripeptides IIIa and IIIb as enniatin analogues.
Preparation of 9-(2-Phosphonomethoxyethyl)adenine Esters as Potential Prodrugs
Authors: Alexander Petr , Holý Antonín , Masojídková Milena
Published: July 2002

Esters of 9-(2-phosphonomethoxyethyl)adenine with substituted aliphatic alcohols were prepared as potential prodrugs. Activation of the phosphonate moiety with dimethylchloromethyleneammonium chloride, generated by reaction of thionyl chloride or triphosgene with dimethylformamide, proved to be the method of choice. The esters were also prepared by alkylation of the phosphonate group with dimethylformamide dialkyl acetals or a mixture of the appropriate alcohol with dimethylformamide dineopentylacetal.
Alkaloids from Papaver pinnatifidum MORIS
Authors: Slavík Jiří , Slavíková Leonora
Published: July 2002

Oripavine (Ia) was isolated from Papaver pinnatifidum MORIS (tetraploid, 2n = 28) of the section Rhoeadium SPACH as the major alkaloid. In a low yield, protopine and (+)-isocorydine were obtained together with a novel levorotatory alkaloid PP 1 of the composition C17H19NO3 and m.p. 179 °C, probably of a normorphinane type. Small amounts of thebaine, allocryptopine, scoulerine, isoboldine, and papaverrubines A, C and D, berberine, and corytuberine were also detected by thin layer chromatography.
2α-Alkoxymethyl Cephalosporins: Reactions of exo-2-Methylene Cephalosporin Sulfones with Alcohols
Authors: Cummings Maxwell D. , Czajkowski David P. , Brunstein John , Reddy Narender A. V. , Phillips Oludotun A. , Fiakpui Charles , Spevak Paul , Micetich Ronald G. , Maiti Samarendra N.
Published: July 2002

A series of 2α-alkoxymethyl cephem sulfones were prepared by nucleophilic addition of a variety of alcohols to exo-2-methylene cephem sulfones. The 2α-alkoxymethyl group was introduced with the aim of improving the inhibitory activity against human leukocyte elastase (HLE) over the unsubstituted compounds. However, against HLE the in vitro activity was still inferior to that shown by the C-2 unsubstituted cephem sulfones.
E-Homolupane Derivatives Substituted in Position 17 and 22a. 1H NMR, 13C NMR and IR Spectra
Authors: Křeček Václav , Hilgard Stanislav , Buděšínský Miloš , Vystrčil Alois
Published: July 2002

A series of derivatives with various oxygen functionalities in positions 17,22a or 19,20 was prepared from diene I and olefin XVI by addition and oxidation reactions. The structure of the obtained compounds was confirmed by 1H NMR, 13C NMR and IR spectroscopy. The kind of intramolecular association of the 17α-hydroxy group was studied in connection with modification of the side chain and substitution in position 22a. Complete assignment of the hydrogen signals and most of the coupling constants was accomplished using a combination of 1D and 2D NMR techniques. The 1H and 13C NMR spectra are discussed.
Reaction of Ester Ethoxycarbonylhydrazones with Aliphatic Diamines
Authors: Ikizler Aykut A. , Sancak Kemal
Published: July 2002

Reaction of esters of (N-ethoxycarbonyl)alkanehydrazonic acids with aliphatic diamines were prepared α,ω-bis(3-alkyl-4,5-dihydro-1H -1,2,4-triazol-5- one-4-yl)alkanes. All compounds were characterized by elemental analyses, IR and 1H NMR spectra.
Jahn-Teller Effect in Hexahydroxocuprate(II) Complexes
Authors: Breza Martin
Published: July 2002

Using semiempirical CNDO-UHF method the adiabatic potential surface of 2[Cu(OH)6]4- complexes is investigated. The values of vibration and vibronic constants for Eg - (a1g + eg) vibronic interaction attain extremal values for the optimal O-H distance. The Jahn-Teller distortion decreases with increasing O-H distance. The discrepancy between experimentally observed elongated bipyramid of [Cu(OH)6]4- in Ba2[Cu(OH)6] and the compressed one obtained by quantum-chemical calculation is explainable by hydrogen bonding of the axial hydroxyl group.
Surface Interactions in Pd-Nb System and Their Relation to Basic Questions and Practical Catalysis
Authors: Jirsak Tomas , Nikolajenko Vladimír , Knor Zlatko
Published: July 2002

Room temperature adsorption-desorption properties of Pd overlayers of various thicknesses were studied by thermal desorption spectroscopy of carbon monoxide. Pd overlayers were deposited on clean and oxidized Nb foil (NbOx/Nb). Adsorption properties of Pd overlayers deposited on clean Nb at temperature 350-400 K were not influenced by annealing in the temperature range 400 K to 600 K. Annealing above this temperature caused the decrease in the area of the Pd surface. It became probably smoother, exhibiting lower number of adsorption sites for CO. Non-annealed Pd overlayers deposited on the oxide carrier (NbOx/Nb) at temperatures between 350 and 400 K possessed the same adsorption capability as Pd deposited directly on Nb. Pd atoms thus neither massively penetrated into the oxide layer nor were encapsulated by NbOx.
Chemometric Analysis of Substituent Effects. XI. Solvent Effects on Dissociation of 2,6-Disubstituted Benzoic Acids
Authors: Kulhánek Jiří , Pytela Oldřich
Published: July 2002

Eleven symmetrically 2,6-disubstituted benzoic acids (with the following substituents: OCH3, OC2H5, OC3H7, OCH(CH3)2, OC4H9, CH3, F, Cl, Br, I, and NO2) have been synthesized and their dissociation constants measured potentiometrically in methanol, ethanol, propan-1-ol, propan-2-ol, butan-2-ol, acetone, dimethyl sulfoxide, dimethylformamide, acetonitrile, pyridine, and 1,2-dichloroethane. The experimental data obtained have been analyzed from the point of view of solvent effects on acidity of the individual derivatives. Different behaviour found with benzoic acid and the disubstituted derivatives in protic solvents is due to changes in solvation. The different character of solvation of benzoic acid and the disubstituted derivatives depends on the type of substitution, being manifested only in 2,6-disubstituted benzoic acids. The graphical analysis has shown a distinct trend in the increase of magnitude of deviation of the point of benzoic acid in the series: propan-2-ol, butan-2-ol, propan-1-ol, ethanol, methanol. This order correlates with the steric demands of carbon chain of the alcohols used. The abnormal behaviour of benzoic acid in the dissociation in these alcohols as compared with that of its 2,6-disubstituted derivatives is due to the different extent of solvation of the reaction centre caused by steric hindrance. Against the expectation, benzoic acid appears to be a weaker acid in protic solvents, whereas its alkoxy derivatives are stronger acids. The solvation also minimizes the inductive effect of alkoxy groups in the symmetrically 2,6-disubstituted derivatives. In aprotic solvents the acidity of 2,6-dialkoxybenzoic acids is also increased, in this case as a result of sterically forced deviation of the reaction centre and/or the substituents out of the plane of benzene ring.
Deactivation of Polymer-Supported Palladium Catalystsin the Hydrogenation of 4-Nitrotoluene
Authors: Fišera Roman , Králik Milan , Annus Julius , Krátky Vieroslav , Zecca Marco , Hronec Milan
Published: July 2002

The deactivation of palladium catalysts supported on commercially available sulfonated polystyrene-divinylbenzene resins (DOWEX) in the hydrogenation of 4-nitrotoluene was studied. Catalysts were prepared from 4 and 8 mole % crosslinked polymers, with particle size between 0.04 and 0.80 mm and palladium contents ranging from 0.25 to 1 wt.% (dry state). The catalytic tests were carried out in a batch reactor vigorously shaken, at 30-50 °C, and 0.25 and 0.5 MPa. The analysis of the conversion curves showed that three factors are mainly responsible for the relatively high deactivation rates: (i) the concentration of hydrogen in the neighbourhood of palladium crystallites, (ii) temperature, (iii) the nanomorphology of the support. Some practical advice concerning the employment of polymeric hydrogenation catalysts and relevant operational conditions can be achieved from the results presented herein.
Gas-Phase Flash Reactions of Diborane, Triborane Carbonyl and Tetraborane with Alkynes
Authors: Fox Mark A. , Greatrex Robert , Greenwood Norman N. , Kirk Martin
Published: July 2002

Several known volatile closo-carboranes (1,5-C2B3H5, 1,6-C2B4H6, 2,4-C2B5H7, 1,10-C2B8H10) and their alkyl derivatives and ten new alkyl derivatives of these carboranes were identified from the thermal gas-phase flash reactions of B2H6, B3H7CO and B4H10 with ethyne, propyne and but-2-yne. In addition, nido-C4B2H6 and its methyl derivatives were obtained from B2H6-alkyne flash reactions. All carboranes found in these high-energy reactions are the most thermally stable isomers of their class. The non-isolable borane {B3H7} is suggested as the initiator in all thermal flash reactions reported here.
Calculation of Orthobaric Densities from Equations of State
Authors: Novák Josef P. , Malijevský Anatol , Cibulka Ivan
Published: July 2002

A numerical method for calculation of densities of saturated vapour and liquid phases, and of saturated vapour pressure using an equation of state is proposed. The method proved to be both robust and efficient using the Redlich-Kwong equation of state for methane. The method can be straightforwardly extended for the case of multicomponent systems.
Adsorption and Oxidation of Thiosulfate on the Platinum Electrode in a Slightly Alkaline Medium
Authors: Loučka Tomáš
Published: July 2002

The aim of this research was to study the oxidation and reduction of the adsorbed thiosulfate on the platinum electrode in a slightly alkaline medium. The adsorption was performed at the open circuit conditions. The reduction of the adsorbed layer in the hydrogen region is slower in a slightly alkaline medium than in acid. The mechanism of reduction and oxidation of adsorbed molecules is probably the same. The nonstationary currents measured in presence of thiosulfates showed that the change in the oxidation number does not take place during the adsorption in the double layer region. In the hydrogen region, thiosulfate replaces the adsorbed hydrogen while beeing reduced. Nonstationary currents at higher concentrations of thiosulfate indicate the presence of more layers on the electrode. Upon reaching higher concentrations of thiosulfate the oxidation reaction takes place between thiosulfate in solution and adsorbed product of its reduction. The open circuit potential of the platinum electrode measured in a thiosulfate solution was 0.780 and 0.783 V against the hydrogen electrode in the same solution.
Evaluation of Heterodimerization Constant from Solid-Liquid and Vapor-Liquid Equilibrium Data
Authors: Malijevská Ivona
Published: July 2002

Activity coefficients obtained from the solid-liquid equilibrium data were used to fit the isobaric vapor-liquid equilibrium data to evaluate the vapor-phase equilibrium constant of heterodimerization of the system propanoic acid-trifluoroethanoic acid. The found hetero-dimerization constant is several times higher than that estimated on the basis of the "double-geometric-mean" rule and its temperature dependence has the form ln KAB = 7 196.7/T - 26.80.