bluuebunny/crossref_metadata_2025 huggingface dataset:
Gamma-Ray Bursts: Accumulating Afterglow Implications, Progenitor Clues, and Prospects
Authors: Mészáros P.
Published: July 2002

Gamma-ray bursts (GRBs) are sudden, intense flashes of gamma rays that, for a few blinding seconds, light up in an otherwise fairly dark gamma-ray sky. They are detected at the rate of about once a day, and while they are on, they outshine every other gamma-ray source in the sky, including the sun. Major advances have been made in the last 3 or 4 years, including the discovery of slowly fading x-ray, optical, and radio afterglows of GRBs, the identification of host galaxies at cosmological distances, and evidence showing that many GRBs are associated with star-forming regions and possibly supernovae. Progress has been made in understanding how the GRB and afterglow radiation arises in terms of a relativistic fireball shock model. These advances have opened new vistas and questions on the nature of the central engine, the identity of their progenitors, the effects of the environment, and their possible gravitational wave, cosmic ray, and neutrino luminosity. The debates on these issues indicate that GRBs remain among the most mysterious puzzles in astrophysics.
Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa
Authors: Lau Hollis , Ferlan Jill T. , Brophy Victoria Hertle , Rosowsky Andre , Sibley Carol Hopkins
Published: July 2002

Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites
Plasmodium falciparum
and
Toxoplasma gondii
. Unfortunately, point mutations in the DHFR gene can confer resistance to inhibitors specific to these pathogens. We have developed a rapid system for testing inhibitors of DHFRs from a variety of parasites. We replaced the DHFR gene from the budding yeast
Saccharomyces cerevisiae
with the DHFR-coding region from humans,
P. falciparum
,
T. gondii
,
Pneumocystis carinii
, and bovine or human-derived
Cryptosporidium parvum
. We studied 84 dicyclic and tricyclic 2,4-diaminopyrimidine derivatives in this heterologous system and identified those most effective against the DHFR enzymes from each of the pathogens. Among these compounds, six tetrahydroquinazolines were effective inhibitors of every strain tested, but they also inhibited the human DHFR and were not selective for the parasites. However, two quinazolines and four tetrahydroquinazolines were both potent and selective inhibitors of the
P. falciparum
DHFR. These compounds show promise for development as antimalarial drugs.
Fourteen-Member Macrolides Suppress Interleukin-8 Production but Do Not Promote Apoptosis of Activated Neutrophils
Authors: Tsuchihashi Yoshiko , Oishi Kazunori , Yoshimine Hiroyuki , Suzuki Shoichi , Kumatori Atsushi , Sunazuka Toshiaki , Omura Satoshi , Matsushima Kouji , Nagatake Tsuyoshi
Published: July 2002

A 14-member macrolide was found to inhibit interleukin-8 (IL-8) synthesis in lipopolysaccharide-stimulated neutrophils but did not accelerate apoptosis in activated neutrophils. These data suggest that 14-member macrolides achieve clinical efficacy for chronic airway diseases partly by suppressing IL-8 production by activated neutrophils, but not by enhancing apoptosis in these cells.
Efficacy of Zidovudine Compared to Stavudine, Both in Combination with Lamivudine and Indinavir, in Human Immunodeficiency Virus-Infected Nucleoside-Experienced Patients with No Prior Exposure to Lamivudine, Stavudine, or Protease Inhibitors (Novavir Trial)
Authors: Joly Véronique , Flandre Philippe , Meiffredy Vincent , Brun-Vezinet Françoise , Gastaut Jean-Albert , Goujard Cécile , Remy Gérard , Descamps Diane , Ruffault Annick , Certain Agnès , Aboulker Jean-Pierre , Yeni for the Novavir Study Group Patrick
Published: July 2002

We compared the efficacy and the toxicity of zidovudine (AZT) versus stavudine (d4T), in combination with lamivudine (3TC) and indinavir, in AZT-, dideoxyinosine (ddI)-, and/or dideoxycytosine (ddC)-experienced patients in a randomized comparative multicenter trial. One hundred seventy human immunodeficiency virus type 1 (HIV-1)-infected patients, who had received AZT, ddI, and/or ddC for at least 6 months but were naive for d4T, 3TC, and protease inhibitors, were randomized to AZT at 250 to 300 mg twice daily, 3TC at 150 mg twice daily, and indinavir at 800 mg every 8 h or to d4T at 40 mg twice daily, 3TC at 150 mg twice daily, and indinavir at 800 mg every 8 h. The primary endpoint was time to virological failure, defined as plasma HIV-1 RNA levels of >5,000 copies/ml after at least 8 weeks of antiretroviral therapy. Additional endpoints were change from baseline in CD4 cell counts, AIDS-defining events and adverse events, and proportion of patients with HIV-1 RNA levels of <500 copies/ml and HIV-1 RNA levels of <50 copies/ml. At week 80, 15 patients in the AZT arm and 14 patients in the d4T arm had reached the primary endpoint, and time to virological failure did not differ between the two arms (P= 0.98). In the d4T and in the AZT arms, 67 and 73% of patients, respectively, had HIV-1 RNA levels of <500 copies/ml (P= 0.50). The median change from baseline in CD4 cell count was 195 × 106and 175 × 106/liter for the d4T- and AZT-containing arms, respectively. The proportions of patients with HIV-1 RNA levels of <50 copies/ml at weeks 8, 16, and 24 were similar in the two arms. The occurrence of serious adverse events was not significantly different between arms. In conclusion, in these patients heavily pretreated with AZT, switching from AZT to d4T when initiating indinavir and 3TC did not bring any additional benefit compared to maintaining AZT.
Effects of Soil and Water Content on Methyl Bromide Oxidation by the Ammonia-Oxidizing Bacterium
Nitrosomonas europaea
Authors: Duddleston Khrystyne N. , Bottomley Peter J. , Porter Angela , Arp Daniel J.
Published: July 2002

Little information exists on the potential of NH
3
-oxidizing bacteria to cooxidize halogenated hydrocarbons in soil. A study was conducted to examine the cooxidation of methyl bromide (MeBr) by an NH
3
-oxidizing bacterium,
Nitrosomonas europaea
, under soil conditions. Soil and its water content modified the availability of NH
4
+
and MeBr and influenced the relative rates of substrate (NH
3
) and cosubstrate (MeBr) oxidations. These observations highlight the complexity associated with characterizing soil cooxidative activities when soil and water interact to differentially affect substrate and cosubstrate availabilities.
Effect of Model Sorptive Phases on Phenanthrene Biodegradation: Different Enrichment Conditions Influence Bioavailability and Selection of Phenanthrene-Degrading Isolates
Authors: Grosser Robert J. , Friedrich Michael , Ward David M. , Inskeep William P.
Published: July 2002

The sorption of organic contaminants by natural organic matter (NOM) often limits substrate bioavailability and is an important factor affecting microbial degradation rates in soils and sediments. We hypothesized that reduced substrate bioavailability might influence which microbial assemblages are responsible for contaminant degradation under enrichment culture conditions. Our primary goal was to characterize enrichments in which different model organic solid phases were used to establish a range of phenanthrene bioavailabilities for soil microorganisms. Phenanthrene sorption coefficients (expressed as log

K
D

values) ranged from 3.0 liters kg
−1
for Amberlite carboxylic acid cation-exchange resin (AMB) to 3.5 liters kg
−1
for Biobeads polyacrylic resin (SM7) and 4.2 liters kg
−1
for Biobeads divinyl benzene resin (SM2). Enrichment cultures were established for control (no sorptive phase), sand, AMB, SM7, and SM2 treatments by using two contaminated soils (from Dover, Ohio, and Libby, Mont.) as the initial inocula. The effects of sorption by model phases on the degradation of phenanthrene were evaluated for numerous transfers in order to obtain stable microbial assemblages representative of sorptive and nonsorptive enrichment cultures and to eliminate the effects of the NOM present in the initial inoculum. Phenanthrene degradation rates were similar for each soil inoculum and ranged from 4 to 5 μmol day
−1
for control and sand treatments to approximately 0.4 μmol day
−1
in the presence of the SM7 sorptive phase. The rates of phenanthrene degradation in the highly sorptive SM2 enrichment culture were insignificant; consequently, stable microbial populations could not be obtained. Bacterial isolates obtained from serial dilutions of enrichment culture samples exhibited significant differences in rates of phenanthrene degradation performed in the presence of SM7, suggesting that enrichments performed in the presence of a sorptive phase selected for different microbial assemblages than control treatments containing solid phase phenanthrene.
Genotyping
Cryptosporidium parvum
with an
hsp70
Single-Nucleotide Polymorphism Microarray
Authors: Straub Timothy M. , Daly Don S. , Wunshel Sharon , Rochelle Paul A. , DeLeon Ricardo , Chandler Darrell P.
Published: July 2002

We investigated the application of an oligonucleotide microarray to (i) specifically detect
Cryptosporidium
spp., (ii) differentiate between closely related
C. parvum
isolates and
Cryptosporidium
species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the
hsp70
gene of
Cryptosporidium parvum
was constructed. Labeled
hsp70
targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I
C. parvum
isolates (TU502 and UG502), two
C. parvum
genotype II isolates (Iowa and GCH1), and DNAs from 22 non-
Cryptosporidium
sp. organisms were used to test method specificity. Only DNAs from
C. parvum
isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.
Use of Formalin-Fixed, Propidium Iodide-Stained Human Leukocytes as a Standard for Enumerating CD4
+
T Lymphocytes in a Single-Platform Assay
Authors: Harrison G. M. , Bennett A. J. , Moody M. , Read G. F. , Williams P. E.
Published: July 2002

A new, inexpensive method is described that enables lymphocytes to be enumerated very precisely. Normal leukocytes were simultaneously stained and fixed with a propidium iodide-paraformaldehyde solution. The preparation obtained (CellBeads) was used as an internal standard for cell enumeration by flow cytometry and was stable at 4°C for at least 60 days. Unlike synthetic beads, the CellBeads behaved similarly to normal cells during red blood cell lysis and cell washing procedures. When known numbers of CellBeads were added to whole-blood samples and the numbers of CellBeads and lymphocytes were determined, highly reproducible and accurate enumerations were obtained—far more so than when synthetic beads were used. This inexpensive method is suitable for routine use.
Cell-Mediated Immune Response during PrimaryChlamydia pneumoniaeInfection
Authors: Halme S. , Latvala J. , Karttunen R. , Palatsi I. , Saikku P. , Surcel H.-M.
Published: July 2002

The development ofChlamydia pneumoniae-specific cell-mediated immunity was studied during a primaryC. pneumoniaeinfection. The immune response was detected as positive lymphocyte proliferation and secretion of interferon gamma.C. pneumoniae-induced activation of both CD4+and CD8+T cells was detected in the early phase of infection, but activation of only CD4+T cells was detected in the later stage.
Antigen Specificity in Experimental Bovine Tuberculosis
Authors: Rhodes S. G. , Gavier-Widen D. , Buddle B. M. , Whelan A. O. , Singh M. , Hewinson R. G. , Vordermeier H. M.
Published: July 2002

This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with
Mycobacterium bovis
. Increased antigen-specific lymphocyte proliferation, gamma interferon, and interleukin-2 responses were observed in all calves following infection. Positive lymphocyte proliferation and cytokine responses to PPD-M and ESAT-6 were observed throughout the infection period studied. In contrast, responses to all other antigens were more variable and were not constantly present, suggesting that antigen cocktails rather than individual antigens should be used for immunodiagnosis. The detection of cytokine responses in the absence of lymphocyte proliferation, particularly during the early stages of infection, suggests a role for antigen-specific cytokine readout systems in the early identification of
M. bovis
infection in cattle.
Activity and Cross-Reactivity of Antibodies Induced in Mice by Immunization with a Group B Meningococcal Conjugate
Authors: Coquillat D. , Bruge J. , Danve B. , Latour M. , Hurpin C. , Schulz D. , Durbec P. , Rougon G.
Published: July 2002

The capsular polysaccharide of group BNeisseriameningitidisis composed of a linear homopolymer of α(2-8)N-acetyl neuraminic acid or polysialic acid (PSA) that is also carried by isoforms of the mammalian neural cell adhesion molecule (NCAM), which is especially expressed on brain cells during development. Here we analyzed the ability of antibodies induced by the candidate vaccineN-propionyl polysaccharide tetanus toxoid conjugate to recognize PSA-NCAM. We hyperimmunized mice to produce a pool of antisera and a series of immunoglobulin G monoclonal antibodies and evaluated their self-reactivity profile by using a battery of tests (immunoprecipitation, immunoblotting, and immunofluorescence detection on live cells and human tissue sections) chosen for their sensitivity and specificity to detect PSA-NCAM in various environments. We also searched for the effects of the vaccine-induced antibodies in two functional assays involving cell lysis or cell migration. Although they were highly bactericidal, all the antibodies tested showed very low or no recognition of PSA-NCAM, in contrast to PSA-specific monoclonal antibodies used as controls. Different patterns of cross-reactions were revealed by the tests used, likely due to affinity and specificity differences among the populations of induced antibodies. Furthermore, neither cell lysis nor perturbation of migration was observed in the presence of the tested antibodies. Importantly, we showed that whereas enzymatic removal of PSA groups from the surfaces of live cells perturbed their migration, blocking them with PSA-specific antibodies was not functionally detrimental. Taken together, our data indicated that this candidate vaccine induced antibodies that could not demonstrate an immunopathologic effect.
Oral Administration of Formaldehyde-Killed Recombinant Bacteria Expressing a Mimic of the Shiga Toxin Receptor Protects Mice from Fatal Challenge with Shiga-Toxigenic
Escherichia coli
Authors: Paton James C. , Rogers Trisha J. , Morona Renato , Paton Adrienne W.
Published: July 2002

Gastrointestinal disease caused by Shiga toxin-producing
Escherichia coli
(STEC) is frequently complicated by life-threatening toxin-induced systemic sequelae, including the hemolytic uremic syndrome. We previously constructed a recombinant bacterium displaying a Shiga toxin receptor mimic on its surface which neutralized Shiga toxins with very high efficiency. Moreover, oral administration of the live bacterium completely protected mice from challenge with virulent STEC. In this study, we investigated the protective capacity of formaldehyde-killed receptor mimic bacteria, as these are likely to be safer for administration to humans. The killed bacteria completely protected STEC-challenged mice when administered three times daily; incomplete protection was achieved using two doses per day. Commencement of therapy could be delayed for up to 48 h after challenge without diminishing protection, depending on the virulence of the challenge strain. Thus, administration of this agent early in the course of human STEC disease may prevent progression to life-threatening complications.
Functional Opsonic Activity of Human Serum Antibodies to Inner Core Lipopolysaccharide (galE) of Serogroup B Meningococci Measured by Flow Cytometry
Authors: Plested Joyce S. , Ferry Berne L. , Coull Philip A. , Makepeace Katherine , Lehmann Anne K. , MacKinnon Fiona G. , Griffiths Helen G. , Herbert Mark A. , Richards James C. , Moxon E. Richard
Published: July 2002

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) ofNeisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixedN. meningitidisorganisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) ofN. meningitidisstrain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope ofN. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r2= 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r2= 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against theN. meningitidisMC58galEmutant in the presence of human complement (r2= 0.994,P= 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.
Magnesium Uptake by CorA Is Essential for Viability of the Gastric Pathogen
Helicobacter pylori
Authors: Pfeiffer Jens , Guhl Johannes , Waidner Barbara , Kist Manfred , Bereswill Stefan
Published: July 2002

We show here that Mg
2+
acquisition by CorA is essential for
Helicobacter pylori
in vitro, as
corA
mutants did not grow in media without Mg
2+
supplementation. Complementation analysis performed with an
Escherichia coli corA
mutant revealed that
H. pylori
CorA transports nickel and cobalt in addition to Mg
2+
. However, Mg
2+
is the dominant CorA substrate, as the
corA
mutation affected neither cobalt and nickel resistance nor nickel induction of urease in
H. pylori
. The drastic Mg
2+
requirement (20 mM) of
H. pylori corA
mutants indicates that CorA plays a key role in the adaptation to the low-Mg
2+
conditions predominant in the gastric environment.
The TRAP-Like SplA Protein Is a
trans
-Acting Negative Regulator of Spore Photoproduct Lyase Synthesis during
Bacillus subtilis
Sporulation
Authors: Fajardo-Cavazos Patricia , Nicholson Wayne L.
Published: July 2002

UV resistance of bacterial endospores derives from a unique DNA photochemistry in which the major UV photoproduct is the thymine dimer 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]) instead of cyclobutane pyrimidine dimers. Repair of SP during spore germination is due in large part to the activity of the enzyme SP lyase encoded by
splB
, the second cistron of the
splAB
operon. Expression of the
splAB
operon in
Bacillus subtilis
is transcriptionally activated by the Eς
G
form of RNA polymerase during morphological stage III in the developing forespore compartment, and SP lyase is packaged into the dormant spore. In addition to temporal and compartmental control of
splAB
expression, a second regulatory circuit which modulates the level of expression of
splB-lacZ
fusions without altering their developmental timing or compartmentalization is reported here. This second regulatory circuit involves the negative action of the
splA
gene product, a 79-amino-acid protein with approximately 50% similarity and 17% identity to TRAP, the tryptophan RNA-binding attenuation protein from
B. subtilis
and
Bacillus pumilus
.
Control of
asgE
Expression during Growth and Development of
Myxococcus xanthus
Authors: Garza Anthony G. , Harris Baruch Z. , Greenberg Brandon M. , Singer Mitchell
Published: July 2002

One of the earliest events in the
Myxococcus xanthus
developmental cycle is production of an extracellular cell density signal called A-signal (or A-factor). Previously, we showed that cells carrying an insertion in the
asgE
gene fail to produce normal levels of this cell-cell signal. In this study we found that expression of
asgE
is growth phase regulated and developmentally regulated. Several lines of evidence indicate that
asgE
is cotranscribed with an upstream gene during development. Using primer extension analyses, we identified two 5′ ends for this developmental transcript. The DNA sequence upstream of one 5′ end has similarity to the promoter regions of several genes that are A-signal dependent, whereas sequences located upstream of the second 5′ end show similarity to promoter elements identified for genes that are C-signal dependent. Consistent with this result is our finding that mutants failing to produce A-signal or C-signal are defective for developmental expression of
asgE
. In contrast to developing cells, the large majority of the
asgE
transcript found in vegetative cells appears to be monocistronic. This finding suggests that
asgE
uses different promoters for expression during vegetative growth and development. Growth phase regulation of
asgE
is abolished in a
relA
mutant, indicating that this vegetative promoter is induced by starvation. The data presented here, in combination with our previous results, indicate that the level of AsgE in vegetative cells is sufficient for this protein to carry out its function during development.
sal
Genes Determining the Catabolism of Salicylate Esters Are Part of a Supraoperonic Cluster of Catabolic Genes in
Acinetobacter
sp. Strain ADP1
Authors: Jones Rheinallt M. , Pagmantidis Vassilis , Williams Peter A.
Published: July 2002

A 5-kbp region upstream of the
are-ben-cat
genes was cloned from
Acinetobacter
sp. strain ADP1, extending the supraoperonic cluster of catabolic genes to 30 kbp. Four open reading frames,
salA
,
salR
,
salE
, and
salD
, were identified from the nucleotide sequence. Reverse transcription-PCR studies suggested that these open reading frames are organized into two convergent transcription units,
salAR
and
salDE
. The
salE
gene, encoding a protein of 239 residues, was ligated into expression vector pET5a. Its product, SalE, was shown to have esterase activity against short-chain alkyl esters of 4-nitrophenol but was also able to hydrolyze ethyl salicylate to ethanol and salicylic acid. A mutant of ADP1 with a Km
r
cassette introduced into
salE
had lost the ability to utilize only ethyl and methyl salicylates of the esters tested as sole carbon sources, and no esterase activity against ethyl salicylate could be detected in cell extracts. SalE was induced during growth on ethyl salicylate but not during growth on salicylate itself.
salD
encoded a protein of undetermined function with homologies to the
Escherichia coli
FadL membrane protein, which is involved in facilitating fatty acid transport, and a number of other proteins detected during aromatic catabolism, which may also function in hydrocarbon transport or uptake processes. A Km
r
cassette insertion in
salD
deleteriously affected cell growth and viability. The
salA
and
salR
gene products closely resemble two
Pseudomonas
proteins, NahG and NahR, respectively encoding salicylate hydroxylase and the LysR family regulator of both salicylate and naphthalene catabolism.
salA
was cloned into pUC18 together with
salR
and
salE
, and its gene product showed salicylate-inducible hydroxylase activity against a range of substituted salicylates, with the same relative specific activities as found in wild-type ADP1 grown on salicylate. Mutations involving insertion of Km
r
cassettes into
salA
and
salR
eliminated expression of salicylate hydroxylase activity and the ability to grow on either salicylate or ethyl salicylate. Studies of mutants with disruptions of genes of the β-ketoadipate pathway with or without an additional
salE
mutation confirmed that ethyl salicylate and salicylate were channeled into the β-ketoadipate pathway at the level of catechol and thence dissimilated by the
cat
gene products. SalR appeared to regulate expression of
salA
but not
salE
.
A ς
54
Activator Protein Necessary for Spore Differentiation within the Fruiting Body of
Myxococcus xanthus
Authors: Gorski Lisa , Gronewold Thomas , Kaiser Dale
Published: July 2002

Insertion of an internal DNA fragment into the
act1
gene, which encodes one of several ς
54
-activator proteins in
Myxococcus xanthus
, produced a mutant defective in fruiting body development. While fruiting-body aggregation appears normal in the mutant, it fails to sporulate (<10
−6
the wild-type number of viable spores). The A and C intercellular signals, which are required for sporulation, are produced by the mutant. But, while it produces A-factor at levels as high as that of the wild type, the mutant produces much less C-signal than normal, as measured either by C-factor bioassay or by the total amount of C-factor protein detected with specific antibody. Expression of three C-factor-dependent reporters is altered in the mutant: the level of expression of Ω4414 is about 15% of normal, and Ω4459 and Ω4403 have alterations in their time course. Finally, the methylation of FrzCD protein is below normal in the mutant. It is proposed that Act1 protein responds to C-signal reception by increasing the expression of the
csgA
gene. This C-signal-dependent increase constitutes a positive feedback in the wild type. The
act1
mutant, unable to raise the level of
csgA
expression, carries out only those developmental steps for which a low level of C-signaling is adequate.
Symbiotic Plasmid Rearrangement in
Rhizobium leguminosarum
bv. viciae VF39SM
Authors: Zhang Xue-Xian , Kosier Bob , Priefer Ursula B.
Published: July 2002

A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in
Rhizobium leguminosarum
bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to
nod, fix
, and
nif
genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.
Identification of a Protein That Inactivates the Competence-Stimulating Peptide of
Streptococcus pneumoniae
Authors: Bergé Mathieu , Langen Hanno , Claverys Jean-Pierre , Martin Bernard
Published: July 2002

Competence for genetic transformation of
Streptococcus pneumoniae
is a transient physiological property inducible by a competence-stimulating peptide (CSP). A 68-kDa CSP-inactivating protein was previously obtained following lithium chloride (LiCl) extraction. By the same protocol, a CSP-inactivating protein was purified and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry as an endopeptidase, PepO. Analysis of a
pepO
mutant provided no support for the hypothesis that PepO participates in competence regulation. To reconcile in vitro and in vivo data, we suggest that LiCl treatment results in the release of intracellular molecules, including PepO.
Comparative Performance of Herpes Simplex Virus Type 2-Specific Serologic Assays from Meridian Diagnostics and MRL Diagnostics
Authors: Ribes Julie A. , Hayes Marie , Smith Angela , Winters Jeffrey L. , Baker Doris J.
Published: July 2002

MRL Diagnostics and Meridian Diagnostics have recently designed herpes simplex virus type 2 (HSV-2)-specific enzyme immunoassays for HSV-2 antibody detection. Blood donor sera were assayed for HSV-2 antibodies by both methods. The sensitivity, specificity, and efficiency were 97.9, 95.4, and 95.9% for the MRL assay and 83.2, 98.2, and 95.5% for the Meridian assay, respectively.
The Susceptibility of
Mycobacterium tuberculosis
to Isoniazid and the Arg→Leu Mutation at Codon 463 of
katG
Are Not Associated
Authors: van Doorn H. R. , Kuijper E. J. , van der Ende A. , Welten A. G. A. , van Soolingen D. , de Haas P. E. W. , Dankert J.
Published: July 2002

A mutation (CCG→CTG [Arg→Leu]) in codon 463 of
katG
(catalase peroxidase) of
Mycobacterium tuberculosis
has been found in isoniazid (INH)-resistant strains. A PCR restriction endonuclease analysis to detect this mutation was applied to 395
M. tuberculosis
isolates from patients in The Netherlands. The proportion of isolates with a detectable mutation was 32% (32 out of 100) and 29% (85 out of 295) among INH-susceptible isolates and INH-resistant or -intermediate isolates, respectively. Sequencing of five INH-susceptible isolates with such mutations showed that all five had the Arg463Leu mutation. We conclude that the Arg463Leu mutation of
katG
of
M. tuberculosis
is not a reliable indicator of INH resistance.
Chlamydia pneumoniae
Serology: Importance of Methodology in Patients with Coronary Heart Disease and Healthy Individuals
Authors: Schumacher A. , Lerkerød A. B. , Seljeflot I. , Sommervoll L. , Holme I. , Otterstad J. E. , Arnesen H.
Published: July 2002

Most publications on the relationship between infection with
Chlamydia pneumoniae
and coronary heart disease (CHD) propose an association, but negative studies are also reported. Seroepidemiological studies vary in the use of different serological methods, different cutoff limits, different sampling times in relation to acute cardiac events, and different clinical stages of CHD. We wanted to compare three different commercially available methods for measuring
Chlamydia
antibodies to see how the choice of method influenced the prevalence of seropositive individuals in CHD patients and in healthy individuals and to see if sampling time in relation to an acute cardiac event or the stage of atherothrombotic disease influenced the results. Blood samples from 197 CHD patients and 197 individually matched healthy control individuals were tested at baseline and after 6 months; the mean age was 55 years in both groups, and 18% were women. Among the CHD patients, 166 were included at a median of 16 days after an acute cardiac event and 31 had chronic disease with the latest acute event being >3 months earlier. The difference in prevalence of antibodies between the CHD patients and the healthy controls was significant when
Chlamydia
lipopolysaccharide antibodies were measured, while no significant differences between the study groups were observed by the two methods detecting
Chlamydia pneumoniae
major outer membrane protein antibodies. The number of seropositive individuals was quite similar at inclusion and 6 months later, and no significant differences were observed between patients with a recent cardiac event and those with a more remote cardiac event. We conclude that the choice of serological method is of major importance when evaluating a possible relationship between
C. pneumoniae
and CHD.
CD28 Costimulatory Blockade Exacerbates Disease Severity and Accelerates Epitope Spreading in a Virus-Induced Autoimmune Disease
Authors: Neville Katherine L. , Dal Canto Mauro C. , Bluestone Jeffrey A. , Miller Stephen D.
Published: July 2002

Theiler's murine encephalomyelitis virus (TMEV) is a natural mouse pathogen which causes a lifelong persistent infection of the central nervous system (CNS) accompanied by T-cell-mediated myelin destruction leading to chronic, progressive hind limb paralysis. TMEV-induced demyelinating disease (TMEV-IDD) is considered to be a highly relevant animal model for the human autoimmune disease multiple sclerosis (MS), which is thought to be initiated as a secondary consequence of a virus infection. Although TMEV-IDD is initiated by virus-specific CD4+T cells targeting CNS-persistent virus, CD4+T-cell responses against self myelin protein epitopes activated via epitope spreading contribute to chronic disease pathogenesis. We thus examined the ability of antibodies directed against B7 costimulatory molecules to regulate this chronic virus-induced immunopathologic process. Contrary to previous studies showing that blockade of B7-CD28 costimulatory interactions inhibit the initiation of experimental autoimmune encephalomyelitis, treatment of SJL mice at the time of TMEV infection with murine CTLA-4 immunoglobulin or a combination of anti-B7-1 and anti-B7-2 antibodies significantly enhanced clinical disease severity. Costimulatory blockade inhibited early TMEV-specific T-cell and antibody responses critical in clearing peripheral virus infection. The inhibition of virus-specific immune responses led to significantly increased CNS viral titers resulting in increased damage to myelin-producing oligodendrocytes. Following clearance of the costimulatory antagonists, epitope spreading to myelin epitopes was accelerated as a result of the increased availability of myelin epitopes leading to a more severe chronic disease course. Our results raise concern about the potential use of B7-CD28 costimulatory blockade to treat human autoimmune diseases potentially associated with acute or persistent virus infections.
Human Immunodeficiency Virus Type 1 Envelope Epitope-Specific CD4+T Lymphocytes in Simian/Human Immunodeficiency Virus-Infected and Vaccinated Rhesus Monkeys Detected Using a Peptide-Major Histocompatibility Complex Class II Tetramer
Authors: Kuroda Marcelo J. , Schmitz Jörn E. , Lekutis Christine , Nickerson Christine E. , Lifton Michelle A. , Franchini Genoveffa , Harouse Janet M. , Cheng-Mayer Cecilia , Letvin Norman L.
Published: July 2002

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4+T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4+T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infectedMamu-DR*W201+rhesus monkeys failed to demonstrate tetramer-binding CD4+T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4+T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201+monkeys vaccinated with a recombinant canarypox virus–HIV-1envconstruct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4+T cells. Thus, Env p46-specific CD4+T cells can be detected by tetrameric Mamu-DR*W201–p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.
Functional Mapping of Protective Domains and Epitopes in the Rotavirus VP6 Protein
Authors: Choi Anthony H. C. , Basu Mitali , McNeal Monica M. , Flint Jason , VanCott John L. , Clements John D. , Ward Richard L.
Published: July 2002

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed inEscherichia coliwas examined. Mice (BALB/c) immunized with two 9-μg doses of each of the chimeras and 10 μg of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively;P≤ 0.01) following challenge. Because CD produced almost complete protection, we prepared fourE. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P≤ 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-μg doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P≤ 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4+T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4+T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4+T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.
Processing and Presentation of Murine Cytomegalovirus pORFm164-Derived Peptide in Fibroblasts in the Face of All Viral Immunosubversive Early Gene Functions
Authors: Holtappels Rafaela , Grzimek Natascha K. A. , Simon Christian O. , Thomas Doris , Dreis Doris , Reddehase Matthias J.
Published: July 2002

CD8 T cells are the principal effector cells in the resolution of acute murine cytomegalovirus (mCMV) infection in host organs. This undoubted antiviral and protective in vivo function of CD8 T cells appeared to be inconsistent with immunosubversive strategies of the virus effected by early (E)-phase genesm04,m06, andm152. The so-called immune evasion proteins gp34, gp48, and gp37/40, respectively, were found to interfere with peptide presentation at different steps in the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation in fibroblasts. Accordingly, they were proposed to prevent recognition and lysis of infected fibroblasts by cytolytic T lymphocytes (CTL) during the E phase of viral gene expression. We document here that the previously identified MHC class I Dd-restricted antigenic peptide257AGPPRYSRI265encoded by genem164is processed as well as presented for recognition by m164-specific CTL during the E and late phases of viral replication in the very same cells in which the immunosubversive viral proteins are effectual in preventing the presentation of processed immediate-early 1 (m123-exon 4) peptide168YPHFMPTNL176. Thus, while immunosubversion is a reality, these mechanisms are apparently not as efficient as the term immune evasion implies. The pORFm164-derived peptide is the first noted peptide that constitutively escapes the immunosubversive viral functions. The most important consequence is that even the concerted action of all immunosubversive E-phase proteins eventually fails to prevent immune recognition in the E phase. The bottom-line message is that there exists no immune evasion of mCMV in fibroblasts.
1H NMR Conformational Studies in Water of Angiotensin II Analogues Modified at the N- and C-Termini: Interactions of the Aromatic Side Chains and Folding of the N-Terminal Domain
Authors: Hondrelis John , Matsoukas John , Agelis George , Cordopatis Paul , Zhou Ning , Vogel Hans , Moore Graham J.
Published: July 2002

The conformation of [Sar1]angiotensin II in water at neutral pH has been examined by proton magnetic resonance spectroscopy at 400 MHz and in particular by comparing its 1H NMR spectral data with those of analogues modified at positions 1,4 and 6, namely [Sar1,Cha8]ANGII, [Des Asp1,Cha8]ANGII, [Aib1,Tyr(Me)4]ANGII, [Aib1,Tyr(Me)4,Ile8]ANGII, [N-MeAib1,Tyr(Me)4]ANGII, [N-MeAib1,Tyr(Me)4,Ile8]ANGII, ANGIII and [Sar1,Ile8]ANGII. Assignment of all proton resonances in these analogues was made possible by 2D COSY NMR experiments. The H-2 and H-4 protons for the histidine ring in [Sar1]ANGII, ANGII and ANGIII were shielded compared with the same protons in [Sar1,Ile8]ANGII, [Sar1,Cha8]ANGII and [Des Asp1,Cha8]ANGII; this shielding effect was not disturbed upon methylation of the tyrosine hydroxyl and/or replacement of residue 1 (sarcosine or aspartic acid) with aminoisobutyric acid (Aib) or N-methyl aminoisobutyric acid (N-MeAib). These data are consistent with our previous suggestion based on NMR studies in neutral DMSO that a characteristic folded conformation for ANGII previously observed in non-polar solvents can also be detected in water at neutral pH, but to a lesser degree.
Effect of Heteroatom on Properties of SiO2-Supported Heteropolymolybdates
Authors: Spojakina Alla , Kostova Nina , Jirátová Květa
Published: July 2002

SiO2-supported trihydrogen phosphododecamolybdate acid H3PMo12O40 (denoted as H3PMo) and tetrahydrogen silicododecamolybdate acid H4SiMo12O40 (denoted as H4SiMo) and their lithium salts have been studied using IR spectroscopy, temperature programmed reduction and measurement of the thiophene conversion. Lacunar anions of H3PMo and, under some conditions, of H4SiMo are formed if lithium is present in the catalyst. Unlike the supported hetero polyacids, lithium combined with the analogues of defect Keggin anions formed during calcination enhances hydrodesulfurization of thiophene.
Synthesis of Carba Analogues of Deoxy-4-C-(hydroxymethyl)pentofuranoses, Intermediates in the Synthesis of Carbocyclic Nucleosides
Authors: Hřebabecký Hubert , Masojídková Milena , Holý Antonín
Published: July 2002

Racemic dimethyl 4-methoxy- (11 and 12), diallyl 4-allyloxy- (13 and 14) and dimethyl 4-(ethylsulfanyl)-2-hydroxycyclopentane-1,1-dicarboxylates (15 and 16) were prepared by base-catalyzed addition of methanol, allyl alcohol and ethylsulfane, respectively, to dimethyl (4-oxobut-2-en-1-yl)malonate (6). Deallylation of 13 and 14 afforded 4-hydroxycyclopentanes 27 and 28. Reduction of 11-16 with lithium aluminium hydride gave the corresponding 4-substituted 2,2-bis(hydroxymethyl)cyclopentanols. Dimethyl (2S,3S,4R)-, (2R,3S,4R)-3-benzyloxy-4-formyloxy-2-hydroxycyclopentane-1,1-dicarboxylates (35, 36) and dimethyl (2S,3S,4R)-, (2R,3S,4R)-3-benzyloxy-2-benzoyloxy-4-methoxycyclopentane-1,1-dicarboxylates (39, 40) were synthesized starting from D-glucose. Reduction of dimethyl cyclopentane-1,1-dicarboxylates 39 and 40 with lithium aluminium hydride, benzoylation of the formed hydroxy derivatives, hydrogenolysis of benzyl groups, conversion of the liberated hydroxy groups into dithiocarbonates and their reduction with tributylstannane afforded, after removal of the protecting groups, (2R,4R)-1,1-bis(hydroxymethyl)-4-methoxycyclopentan-2-ol ((2R,4R)-17) and (3R,4R)-1,1-bis(hydroxymethyl)-4-methoxycyclopentan-3-ol (51). Reduction of a mixture of esters 35 and 36 gave (2R,3R)-2-benzyloxy-5-(hydroxymethyl)hexane-1,3,6-triol (52) as the major product and (2R,3S,4R)-3-benzyloxy-1,1-bis(hydroxymethyl)cyclopentane-2,4-diol (53) as the minor product. The latter was converted into (3R,4R)-1,1-bis(hydroxymethyl)cyclopentane-3,4-diol (58). 3-Deoxycarba analogues 51 and 58 arose by migration of benzoyl group in the preparation of the dithiocarbonates.
Steric Effects and Steric Inhibition of Resonance in Isopropylbenzoic Acids in the Gas Phase and in Solution
Authors: Fiedler Pavel , Kulhánek Jiří , Decouzon Michèle , Gal Jean-François , Maria Pierre-Charles , Exner Otto
Published: July 2002

The substituent effects of the isopropyl group in 2-, 3- and 4-isopropylbenzoic acids were investigated on the enthalpies of formation, gas-phase acidities, acidities in methanol and in dimethyl sulfoxide, and on the IR spectra in tetrachloromethane. Particular attention was given to the influence of variable conformation on the observed steric effect. In contrast to 2-tert-butylbenzoic acid and similarly to 2-methylbenzoic acid, 2-isopropylbenzoic acid exists in two planar conformations in equilibrium. Due to this conformational freedom, the steric effects of the isopropyl group on the conformation of the carboxyl group or on the gas-phase acidity are relatively small, rather close to that of a methyl group. The gas-phase acidity can be qualitatively described in terms of pole/induced dipole interaction in the anion. The latter effect is observable with a lower intensity even in 3-isopropylbenzoic acid: in this action from a more remote position, the isopropyl group resembles more the tert-butyl than the methyl group. In methanol and dimethyl sulfoxide solutions, the steric effects and particularly the pole/induced dipole interaction are attenuated and effect of isopropyl acquires intermediate values between methyl and tert-butyl. Solvent effects are thus deciding for the position of the isopropyl substituent on the scale of steric substituent constants.