bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Type 1 Versus Type 2 Immune Responses in Liver During the Onset of Chronic Woodchuck Hepatitis Virus Infection
Authors: Wang Yun , Menne Stephan , Jacob James R. , Tennant Bud C. , Gerin John L. , Cote Paul J.
Published: April 2003

Immune response messenger RNAs (mRNA) were compared in liver during self–limited (resolved) and chronic neonatal woodchuck hepatitis virus (WHV) infection. At week 14 postinfection (mid–acute phase), mRNAs for leukocyte markers (CD3, CD4, CD8), type 1 cytokines and related transcription factors (IFN–γ, TNF–α, STAT4, T–bet), and IL–10 were increased in livers from resolving infections, but mRNAs of other type 1 (IL–2) and type 2 (IL–4, STAT6, and GATA3 markers remained at baseline levels. Increased coexpression of IFN–γ and TNF–α mRNAs correlated in most cases with lower levels of intrahepatic WHV covalently closed circular DNA (cccDNA). At the same time point postinfection, livers from woodchucks that eventually progressed to chronic infection had baseline or slightly elevated levels of CD and type 1 mRNAs, which were significantly lower (or elevated less frequently) compared with resolving woodchucks. Earlier, at week 8, there were no differences between the two outcome settings. During these early time points and at a later stage in chronic infection (15 months), type 2 mRNAs in carrier liver remained at baseline levels or, when elevated, were never in excess of those in resolving woodchucks. In conclusion, the onset and maintenance of neonatal chronic WHV infection are not associated with antagonistic type 2 immunoregulation of type 1 responses in liver. Accordingly, chronicity develops in association with a primary deficiency in the intrahepatic CD responses, especially involving CD8+ T lymphocytes, and in both extracellular (cytokine) and intracellular (transcriptional) type 1 response mediators. This has relevant implications for future treatment of chronic hepatitis B virus (HBV) infection in humans. (Hepatology 2003;37:771–780.)
Effects of pulsatile secretion of growth hormone (GH) on fat deposition in human GH transgenic rats
Authors: Furuhata Yasufumi , Nishihara Masugi , Takahashi Michio
Published: April 2003

Growth hormone (GH) is an endocrine regulator of glucose and lipid metabolism as well as body growth. GH levels are decreased and a unique pulsatile secretory pattern becomes obvious after puberty particularly in males. Coincidentally with this, males tend to deposit body fat. Experimental and clinical evidence has accumulated that obesity is associated with a decrease in GH levels. A strain of transgenic rats has been generated with severe obesity but normal nose-to-tail length, which has low circulating GH levels without pulsatility (human growth hormone (hGH) transgenic rats). The present review mainly focuses on recent and current work analysing the relationship between the occurrence of obesity and low GH levels and/or the absence of GH pulsatility in this transgenic animal model. This model has elevated blood glucose, non-esterified fatty acid, insulin and leptin levels associated with hyperphagia, suggesting that these rats also carry insulin- and leptin-resistant characteristics. hGH transgenic rats were subjected to a pair-feeding treatment to normalize food intake and chronic GH replacement to normalize GH levels. While the pair-feeding for 8 weeks successfully suppressed body-weight gain, the fat pad : body weight ratio remained very similar to freely-eating control hGH transgenic rats, which indicates the hyperphagia is not the sole contributor to the excess fat accumulation in this model. However, continuous elevation of peripheral hGH levels (approximately 2-fold) for 8 weeks by means of a slow-release vehicle resulted in a significant decrease in the fat mass : body weight ratios by 30 %. This GH treatment altered neither food intake nor body-weight gain. Thus, two characteristic phenotypes observed in the hGH transgenic rats, hyperphagia and obesity, seem to be closely related to GH levels and GH secretory pattern. This relationship might be working in the regulation of changes in seasonal body composition in wild animals.
Identification and Characterization of Helicobacter pylori Genes Essential for Gastric Colonization
Authors: Kavermann Holger , Burns Brendan P. , Angermüller Katrin , Odenbreit Stefan , Fischer Wolfgang , Melchers Klaus , Haas Rainer
Published: April 2003

Helicobacter pylori causes one of the most common, chronic bacterial infections and is a primary cause of severe gastric disorders. To unravel the bacterial factors necessary for the process of gastric colonization and pathogenesis, signature tagged mutagenesis (STM) was adapted to H. pylori. The Mongolian gerbil (Meriones unguiculatus) was used as model system to screen a set of 960 STM mutants. This resulted in 47 H. pylori genes, assigned to 9 different functional categories, representing a set of biological functions absolutely essential for gastric colonization, as verified and quantified for many mutants by competition experiments. Identification of previously known colonization factors, such as the urease and motility functions validated this method, but also novel and several hypothetical genes were found. Interestingly, a secreted collagenase, encoded by hp0169, could be identified and functionally verified as a new essential virulence factor for H. pylori stomach colonization. Furthermore, comB4, encoding a putative ATPase being part of a DNA transformation-associated type IV transport system of H. pylori was found to be absolutely essential for colonization, but natural transformation competence was apparently not the essential function. Thus, this first systematic STM application identified a set of previously unknown H. pylori colonization factors and may help to potentiate the development of novel therapies against gastric Helicobacter infections.
Method for Quantitative Detection and Presumptive Identification of Group B Streptococci on Primary Plating
Authors: Hansen Søren Mose , Sørensen Uffe B. Skov
Published: April 2003

Maternal prenatal screening for group B streptococci (GBS) followed by offering of intrapartum chemoprophylaxis to carriers is one of the strategies used to reduce the incidence of neonatal early-onset GBS infections. Culturing of vaginal and anorectal swab specimens in selective broth is the screening procedure recommended by the Centers for Disease Control and Prevention. This technique is sensitive; it does not, however, allow either evaluation of the degree of colonization or detection of cocolonization with several GBS clones. We have examined the carriage rate and population dynamics of GBS in a group of Danish women during pregnancy and 1 year after delivery using a new detection method. In the present paper we describe a mixed blood agar medium (MB agar) that identifies GBS in the primary cultures by detection of a double hemolysis pattern consisting of characteristic, large zones of partial hemolysis (“CAMP zones”) and of narrow zones of complete hemolysis. The MB agar was at least as sensitive as culturing in selective broth for detection of GBS in vaginal and anorectal swab specimens, and GBS strains could be identified directly on the primary plate due to the CAMP zones without the need for subculturing. The carriage rate of GBS in a group of Danish women was found to be more than 30%, a figure considerably higher than the rate that was reported previously.
Development of a Real-Time PCR Assay for Quantitative Detection of
Encephalitozoon intestinalis
DNA
Authors: Menotti Jean , Cassinat Bruno , Sarfati Claudine , Liguory Olivier , Derouin Francis , Molina Jean-Michel
Published: April 2003

A new real-time PCR assay for quantitation of
Encephalitozoon intestinalis
DNA was developed which used a
Taq
Man fluorescent probe for specific detection. Serial dilutions of
E. intestinalis
spore suspensions obtained from tissue culture were used as external standards. The detection limit of the technique was 20 spores per ml, with a good interassay reproducibility (coefficient of variation of 7.1% for the suspension containing 20 spores/ml, 5.0% for the suspension containing 75 spores/ml and below 3.5% for higher concentrations). Quantitative detection of
E. intestinalis
DNA was similar whether the serial dilutions of spores were made in distilled water or in a stool suspension, allowing the use of the assay for stool specimens. The assay was then applied to 14 clinical specimens from 8 immunocompromised patients with proven
E. intestinalis
infection. The quantitation of the parasitic burden was achieved in stools, blood, urine, tissue biopsies, and bronchopulmonary specimens. The highest parasitic burdens were noted in stools, urine, and bronchopulmonary specimens, reaching 10
5
to 10
6
spores/g or ml. Dissemination of the infection was also evidenced in some patients by demonstration of
E. intestinalis
DNA in blood and serum. We conclude that real-time PCR is a valuable tool for quantitation of
E. intestinalis
burden in clinical specimens.
International Collaborative Study To Compare Reverse Transcriptase PCR Assays for Detection and Genotyping of Noroviruses
Authors: Vinjé Jan , Vennema Harry , Maunula Leena , von Bonsdorff Carl-Henrik , Hoehne Marina , Schreier Eckart , Richards Alison , Green Jon , Brown David , Beard Suzanne S. , Monroe Stephan S. , de Bruin Erwin , Svensson Lennart , Koopmans Marion P. G.
Published: April 2003

To allow more rapid and internationally standardized assessment of the spread of noroviruses (previously called Norwalk-like viruses [NLVs]) as important food-borne pathogens, harmonization of methods for their detection is needed. Diagnosis of NLVs in clinical diagnostic laboratories is usually performed by reverse transciptase PCR (RT-PCR) assays. In the present study, the performance of five different RT-PCR assays for the detection of NLVs was evaluated in an international collaborative study by five laboratories in five countries with a coded panel of 91 fecal specimens. The assays were tested for their sensitivity, detection limit, and ease of standardization. In total, NLVs could be detected by at least one RT-PCR assay in 69 (84%) of the samples that originally tested positive. Sensitivity ranged from 52 to 73% overall and from 54 to 100% and 58 to 85% for genogroup I and II viruses, respectively. In all, 64% of the false-negative results were obtained with a set of diluted stools (n= 20) that may have lost quality upon storage. Sensitivity was improved when these samples were excluded from analysis. No one single assay stood out as the best, although the p1 assay demonstrated the most satisfactory overall performance. To promote comparability of data, this assay will be recommended for newly starting groups in future collaborative studies.
Activities of Fluconazole and Voriconazole against 1,586 Recent Clinical Isolates of
Candida
Species Determined by Broth Microdilution, Disk Diffusion, and Etest Methods: Report from The ARTEMIS Global Antifungal Susceptibility Program, 2001
Authors: Pfaller M. A. , Diekema D. J. , Messer S. A. , Boyken L. , Hollis R. J.
Published: April 2003

The ARTEMIS Global Antifungal Susceptibility Program (ARTEMIS Program) was initiated in 2001 to provide focused surveillance of the activities of fluconazole and voriconazole against
Candida
spp. isolated from blood and other normally sterile sites. A total of 1,586 episodes of infection were detected at 61 international study sites. Overall, 57.7% of the infections were due to
Candida albicans
, followed by
C. glabrata
(14.8%),
C. parapsilosis
(12.5%),
C. tropicalis
(9.4%),
C. krusei
(2.7%), and
C. lusitaniae
(1.5%). Isolates of
C. albicans
,
C. parapsilosis
, and
C. tropicalis
were all highly susceptible to fluconazole (for 99% of the isolates the MICs were ≤8 μg/ml). Likewise, 99 to 100% of these species were inhibited by ≤1 μg of voriconazole per ml. Voriconazole was also active against
C. glabrata
(93% of the isolates were susceptible [MICs ≤ 1 μg/ml]) and
C. krusei
(100% of the isolates were susceptible). The agar-based Etest and disk diffusion methods performed well for the testing of both fluconazole and voriconazole compared to the broth microdilution MIC reference method. These observations establish the continued importance of
C. albicans
as a pathogen and the sustained activity of fluconazole and the broad spectrum of activity of voriconazole and will serve as the first-year benchmark for the ARTEMIS Program. Continued surveillance and refinement of broth- and agar-based test methods will help to identify susceptibility trends and improve the laboratory capability for antifungal susceptibility testing.
Molecular Analysis of the 18S rRNA Gene of
Cryptosporidium
Parasites from Patients with or without Human Immunodeficiency Virus Infections Living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam
Authors: Gatei Wangeci , Greensill Julie , Ashford Richard W. , Cuevas Luis E. , Parry Christopher M. , Cunliffe Nigel A. , Beeching Nicholas J. , Hart C. Anthony
Published: April 2003

An 840-bp fragment of the 18S rRNA gene was used to identify
Cryptosporidium
spp. recovered from human immunodeficiency virus (HIV)-infected and -uninfected patients from Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. Initial identification was by Ziehl-Neelsen acid-fast staining. Confirmation was by nested PCR, targeting the most polymorphic region of the 18S rRNA gene. Genotyping was by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing. Among 63 isolates analyzed, four genotypes of
Cryptosporidium
were identified; 75% of the isolates were of the
C
.
parvum
human genotype, while the potentially zoonotic species were of the
C. parvum
bovine genotype (21.7%), the
C
.
meleagridis
genotype (1.6% [one isolate]), and the
C
.
muris
genotype (1.6% [one case]). HIV-infected individuals were more likely to have zoonotic genotypes than the HIV-uninfected individuals. Among the
C. parvum
group, strains clustered distinctly into either human or bovine genotypes regardless of the geographical origin, age, or HIV status of the patients. The intragenotypic variation observed in the
C. parvum
human genotype was extensive compared to that within the
C. parvum
bovine genotype group. The variation within genotypes was conserved in all geographical regions regardless of the patients' HIV status. The extensive diversity within genotypes at the 18S rRNA gene locus may limit its application to phylogenetic analyses.
DNA Fingerprinting ofSalmonella entericasubsp.entericaSerovar Typhimurium with Emphasis on Phage Type DT104 Based on Variable Number of Tandem Repeat Loci
Authors: Lindstedt Bjørn-Arne , Heir Even , Gjernes Elisabet , Kapperud Georg
Published: April 2003

Seventy-eight human and environmental strains ofSalmonella entericasubsp.entericaserovar Typhimurium, as well as 18 isolates of otherSalmonellaserovars and 6 isolates ofEscherichia coli, were subjected to a novel variable number of tandem repeats (VNTR)-based fingerprinting method that showed high discrimination and reproducibility for typing serovar Typhimurium isolates. The method is based on capillary separation of PCR products from fluorescence-labeled VNTR in the serovar Typhimurium genome. The serovar Typhimurium isolates displayed 54 VNTR patterns, and the VNTR assay correctly identified strains from a well-characterized outbreak. Among 37 serovar Typhimurium phage type DT104 isolates, 28 distinct VNTR patterns were found. This VNTR-based method is fast and suitable for complete automation. Our VNTR-based method was capable of high discrimination within the homogeneous serovar Typhimurium DT104 phage type and can be used to trace outbreaks and to monitor DT104 as well as other phage types. The VNTR assay was compared toXbaI pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, integron-cassette profiles and gene PCR ofintI1,qacEΔ1,sulI1, andfloR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.
Development of a Duplex PCR Genotyping Assay for Distinguishing
Clostridium perfringens
Type A Isolates Carrying Chromosomal Enterotoxin (
cpe
) Genes from Those Carrying Plasmid-Borne Enterotoxin (
cpe
) Genes
Authors: Wen Qiyi , Miyamoto Kazuaki , McClane Bruce A.
Published: April 2003

About 5% of
Clostridium perfringens
type A isolates carry the
cpe
gene encoding the
C. perfringens
enterotoxin. Those
cpe
-positive type A isolates are important causes of food-poisoning and non-food-borne cases of diarrheas in humans, as well as certain veterinary cases of diarrhea. Previous studies have determined that the enterotoxigenic type A isolates causing both non-food-borne human gastrointestinal disease and veterinary disease carry their
cpe
genes on plasmids, while the type A isolates causing human food poisoning carry a chromosomal
cpe
gene. The present study reports on the successful development of a duplex PCR assay that can rapidly genotype enterotoxigenic type A isolates (i.e., determine whether those
cpe
-positive isolates carry a chromosomal or a plasmid-borne
cpe
gene). The availability of this rapid
cpe
genotyping assay capable of handling large numbers of samples provides a powerful new investigative tool for diagnostic, epidemiologic, and basic research purposes.
Comparison of Ribotyping and Restriction Enzyme Analysis for Inter- and Intraspecies Discrimination of
Bordetella avium
and
Bordetella hinzii
Authors: Register Karen B. , Sacco Randy E. , Nordholm Gwen E.
Published: April 2003

Bordetella avium
is an avian respiratory disease pathogen responsible for substantial economic losses to the turkey industry. The inability to distinguish isolates has hampered outbreak investigations and prevents a complete understanding of transmission mechanisms. Isolates of
Bordetella hinzii
, often referred to as
B. avium
-like or as
Alcaligenes faecalis
type II prior to 1995, have also been acquired from the respiratory tracts of diseased poultry but are not believed to be pathogenic for birds. Therefore, differentiating between
B. avium
and
B. hinzii
is of importance for veterinary diagnostic laboratories. It was recently reported that both
Pvu
II ribotyping and
Hin
fI/
Dde
I restriction endonuclease analysis (REA) show promise for distinguishing isolates of
B. avium
and
B. hinzii
. Here we compare the ability of these techniques to discern inter- and intraspecies differences. While both approaches distinguished numerous types within a species, only REA was sufficiently discriminatory for routine use as an epidemiologic tool. Both techniques clearly distinguish between
B. avium
and
B. hinzii
, although the results of ribotyping are more easily interpreted. Ribotyping and REA identified numerous, previously unrecognized
B. hinzii
strains from a collection of bordetella isolates, including one acquired from a rabbit. This is the first report of
B. hinzii
isolation from a nonhuman mammalian species. At least some of the newly recognized
B. hinzii
isolates have been previously reported to cause disease in poults, suggesting that the pathogenicity of this agent for poultry should be more rigorously examined.
Detection, Differentiation, and Quantitation of Pathogenic
Leishmania
Organisms by a Fluorescence Resonance Energy Transfer-Based Real-Time PCR Assay
Authors: Schulz Alexandra , Mellenthin Katja , Schönian Gabriele , Fleischer Bernhard , Drosten Christian
Published: April 2003

Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of
Leishmania
organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes
Leishmania
organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of ≥95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The
Leishmania donovani
complex, the
Leishmania brasiliensis
complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0°C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.
International Typing Study of Toxin A-Negative, Toxin B-Positive
Clostridium difficile
Variants
Authors: Johnson Stuart , Sambol Susan P. , Brazier Jon S. , Delmée Michel , Avesani V. , Merrigan Michelle M. , Gerding Dale N.
Published: April 2003

Clinically important strains of
Clostridium difficile
that do not produce toxin A but produce toxin B and are cytotoxic (A
−
/B
+
) have been reported from multiple countries. In order to compare the relatedness of these strains, we typed 23 A
−
/B
+
C. difficile
isolates from the United Kingdom (6 isolates), Belgium (11 isolates), and the United States (6 isolates) by three well-described typing methods. Restriction endonuclease analysis (REA), PCR ribotyping, and serogrouping differentiated 11, 4, and 3 different strain types, respectively. Twenty-one of the 23 A
−
/B
+
variants had a 1.8-kb truncation of the toxin A gene characteristic of toxinotype VIII strains; 20 of the 21 toxinotype VIII-like strains were PCR type 17. PCR type 17 isolates could be differentiated into two separate strain groups by serogrouping and by REA. REA further discriminated these isolates into eight subgroups (REA types). PCR type 17-serogroup F-REA group CF isolates were recovered from all three countries, and one specific REA type, CF4, was recovered from patients with
C. difficile
disease in the United Kingdom and the United States.
C. difficile
A
−
/B
+
variants of apparent clonal origin are widely distributed in Europe and North America.
Rapid Detection of Herpes Simplex Virus and Varicella-Zoster Virus Infections by Real-Time PCR
Authors: Weidmann Manfred , Meyer-König Ursula , Hufert Frank T.
Published: April 2003

The herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus (VZV) can cause life-threatening infections of the central nervous system and lead to severe infections in immunocompromised subjects and newborns. In these cases, rapid diagnosis is crucial. We developed three different real-time PCR assays based on TaqMan chemistry for the LightCycler instrument to detect HSV-1, HSV-2, and VZV. When the TaqMan assays were compared to our in-house nested PCR assays, the test systems had equal sensitivities of ≤10 plasmid copies per assay. When clinical samples were investigated by TaqMan PCR to detect HSV-1, HSV-2, and VZV DNA, 95, 100, and 96% of the samples determined to be positive by nested PCR, respectively, were positive by the real-time PCR assays. The specificities of all PCR assays were almost 100%. Furthermore, the TaqMan PCR assays could be performed within 2.5 h, whereas nested PCR results were available after 9 h. In addition to offering more rapid results, the TaqMan PCR assays appear to be less expensive than nested PCR assays due to less hands-on time. In summary, TaqMan PCR is an excellent alternative to conventional nested PCR assays for the rapid detection of HSV-1, HSV-2, and VZV in clinical samples.
Evolution of a Vancomycin-Intermediate
Staphylococcus aureus
Strain In Vivo: Multiple Changes in the Antibiotic Resistance Phenotypes of a Single Lineage of Methicillin-Resistant
S
.
aureus
under the Impact of Antibiotics Administered for Chemotherapy
Authors: Sieradzki K. , Leski T. , Dick J. , Borio L. , Tomasz A.
Published: April 2003

A number of methicillin-resistant
Staphylococcus aureus
(MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent
S
.
aureus
bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 μg/ml for the initial isolate to an MIC of 8 μg/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates—apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern,
spaA
type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.
Blood Agar and
Mycobacterium tuberculosis
: the End of a Dogma
Authors: Drancourt M. , Carrieri P. , Gévaudan M.-J. , Raoult D.
Published: April 2003

Incidental blood agar-based recovery of
Mycobacterium tuberculosis
led us to further investigate this routine medium for primary isolation and culture of
M. tuberculosis
. Fifteen respiratory tract and eight lymph node Ziehl-Neelsen-positive specimens were inoculated in parallel into tubes containing egg-based medium and 5% sheep blood agar. Colonies appeared sooner on this medium than on the egg-based medium, but this difference was not significant (
P
= 0.11, analysis of variance [ANOVA] test). Further experiments compared the growth of 38 respiratory and lymph node
M. tuberculosis
isolates when subcultured on the two media. After 6 days of incubation, 21 of 38 isolates had grown on blood agar, and the mean number of colonies was significantly greater on blood agar than on the egg-based medium (
P
< 0 0.001, ANOVA test). These results demonstrate that
M. tuberculosis
grows easily on blood agar within 1to 2 weeks, indicating that this basic medium is suitable for laboratory diagnosis of tuberculosis in addition to other media. Laboratories that routinely use prolonged incubations of blood plates, for example, for the recovery of
Bartonella
species, should consider the potential safety implications of encountering this highly infectious pathogen.
Enterobacterial Repetitive Intergenic Consensus 1R PCR Assay for Detection of
Raoultella
sp. Isolates among Strains Identified as
Klebsiella oxytoca
in the Clinical Laboratory
Authors: Granier Sophie A. , Leflon-Guibout Véronique , Goldstein Fred W. , Nicolas-Chanoine Marie-Hélène
Published: April 2003

The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of
Raoultella
and the two genetic groups of
Klebsiella oxytoca
, was applied to 19 clinical isolates identified as
K. oxytoca
. By this method, as confirmed by species-specific gene sequencing, two
Raoultella ornithinolytica
and two unclassifiable
K. oxytoca
isolates were identified.
New PCR Test That Recognizes All Human Prototypes of Enterovirus: Application for Clinical Diagnosis
Authors: Bourlet Thomas , Caro Valerie , Minjolle Sophie , Jusselin Isabelle , Pozzetto Bruno , Crainic Radu , Colimon Ronald
Published: April 2003

We describe a new PCR test (Penter RT-PCR) that recognizes all 64 prototypes of enterovirus. Sixty clinical samples were analyzed in parallel with this Penter RT-PCR and previously described PCR tests: 34 and 32 samples tested positive, respectively. This assay is suitable for use in clinical diagnosis, and its ability to amplify all known serotypes makes it more useful than other consensus PCR tests.
New Method for Typing
Staphylococcus aureus
Strains: Multiple-Locus Variable-Number Tandem Repeat Analysis of Polymorphism and Genetic Relationships of Clinical Isolates
Authors: Sabat Artur , Krzyszton-Russjan Jolanta , Strzalka Wojciech , Filipek Renata , Kosowska Klaudia , Hryniewicz Waleria , Travis James , Potempa Jan
Published: April 2003

The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant
Staphylococcus aureus
. Five staphylococcal VNTR loci (
sdr
,
clfA
,
clfB
,
ssp
, and
spa
) were subjected to analysis, and it was shown that the method allows typing of
S
.
aureus
strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.
Electroreduction of Aromatic Nitro Compounds: Case for Comparison of Information Obtained by Polarography and Voltammetry
Authors: Zuman Petr
Published: April 2003

The difference between the information obtained by d.c. polarography (which is virtually a potentiostatic method) and cyclic voltammetry (CV), where products formed at one potential can affect electrolysis at another potential is discussed. The principle is demonstrated on reduction of nitrobenzenes, where at DME the reduction usually occurs as a strictly four-electron process, whereas in CV the arylhydroxylamines formed react wit an intermediate of the four-electron reduction.
1,2-Bis(azacrown)ethanes: Effect of Bridge Substitution and Ring Size on Formation of Sandwich Complexes with Sodium and Potassium Ions
Authors: Bělohradský Martin , Holý Petr , Koudelka Juraj , Závada Jiří
Published: April 2003

The title problem was investigated potentiometrically using four homologous series of bis(azacrown)s I-IV. A surprisingly large destabilizing effect of bridge substituent was found in the intramolecular sandwich complex formation and ascribed to symmetry violation.
Dipole Moments of meta and para Substituted Benzoyl Isothiocyanates
Authors: Danihel Ivan , Suchár Gejza
Published: April 2003

For a series of meta and para substituted benzoyl isothiocyanates, the dipole moments were determined experimentally and calculated by vector addition of the bond and group dipole moments. Confirmation analysis revealed the preference of the synclinal conformation (τ ∀ 60°), which emerges from the rotation about the C(7)-N bond. MNDO calculations were also performed for the compounds, and the dipole moments obtained were compared with those from the above procedure.
Synthesis of 1-(3-Azido-2,3-dideoxy-4-C-hydroxymethyl-α-L-threo-pentofuranosyl)thymine, 1-(2,3-Dideoxy-4-C-hydroxymethyl-α-L-glycero-pentofuranosyl)thymine and 1-(2,3-Dideoxy-4-C-hydroxymethyl-α-L-glycero-pent-2-enofuranosyl)thymine
Authors: Hřebabecký Hubert , Holý Antonín
Published: April 2003

1-(2-O-Acetyl-3,5-di-O-benzoyl-4-C-benzoyloxymethyl-α-L-arabinofuranosyl)thymine (II) was converted to 2,2'-anhydro derivative V by selective deacetylation, mesylation and treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene. Cleavage of 2,2'-anhydro ring in compound V with hydrogen chloride or bromide afforded the respective 2'-chloro and 2'-bromo derivatives VI and VII. Reaction of compound VII with Cu/Zn couple and subsequent debenzoylation led to 1-(2,3-dideoxy-4-C-hydroxymethyl-α-L-pent-2-enofuranosyl)thymine (IX). Catalytic hydrogenation of IX gave 1-(2,3-dideoxy-4-C-hydroxymethyl-α-L-glycero-pentofuranosyl)thymine (X). Dehalogenation of compound VI with tributyltin hydride and debenzoylation afforded 1-(2-deoxy-4-C-hydroxymethyl-α-L-erythro-pentofuranosyl)thymine (XII). Tritylation of compound XII, followed by mesylation, detritylation and nucleophilic substitution with azide furnished 1-(3-azido-2,3-dideoxy-4-C-hydroxymethyl-α-L-threo-pentofuranosyl)thymine (XXII).
Preparation of Purine 2'-Deoxy-5'-O-phosphonomethylnucleosides and 2'-Deoxy-3'-O-phosphonomethylnucleosides
Authors: Krečmerová Marcela , Hřebabecký Hubert , Masojídková Milena , Holý Antonín
Published: April 2003

Sodium salt of 2'-deoxy-N6-dimethylaminomethylene-3'-O-(tetrahydro-2H-pyran-2-yl)adenosine (VIII) reacted with dibenzyl p-toluenesulfonyloxymethanephosphonate (Ia) to give dibenzyl ester of 2'-deoxy-N6-dimethylaminomethylene-5'-O-phosphonomethyl-3'-O-(tetrahydro-2H-pyran-2-yl)adenosine (XI) which after deprotection afforded the final 2'-deoxy-5'-O-phosphonomethyladenosine (XII). 2'-Deoxy-5'-O-hydroxymethanephosphonyladenosine (XIV) and 5'-O-benzyloxymethanephosphonyl-2'-deoxyadenosine (XIII) were isolated as a side product. The preparation of 2'-deoxy-5'-O-phosphonomethylguanosine (XVI) and protection of the starting nucleoside were analogous to those of compound XII. In the 2'-deoxy-3'-O-phosphonomethylnucleosides series, 2'-deoxy-3'-O-phosphonomethylcytidine (XXI) and 2'-deoxy-3'-O-phosphonomethyladenosine (XXVII) were prepared, using N4-benzoyl-5'-O-tert-butyldiphenylsilyl-2'-deoxycytidine (XVIII) and 5'-O-tert-butyldiphenylsilyl-2'-deoxy-N6-dimethylaminomethyleneadenosine (XXIV), respectively, as starting compounds.
World of van der Waals Species
Authors: Hobza Pavel , Zahradník Rudolf
Published: April 2003

Van der Waals species (molecules, ions, radicals) attract the attention of chemists, chemical physicists and molecular biologists. Study of these systems presents a real challenge for both experimentalists and theorists. The main features of the contemporary possibilities for quantum chemical treatment are illustrated mostly on systems studied in the authors' laboratory. Prospects in the area are briefly outlined.
Nucleophilic Vinylic Substitution on α-Tosyloxymethylene Lactones
Authors: Mazal Ctibor , Jonas Jaroslav
Published: April 2003

Sodium salt of 3-hydroxymethylenetetrahydro-2H-pyran-2-one (V), obtained by Claisen condensation of δ-valerolactone with ethyl formate, was converted into its sulfonates and carboxylates IV, VII - X, which were obtained either as pure E-isomers or as mixtures of E- and Z-isomers; the mixtures were chromatographically separated. Substitution reaction of α-tosyloxymethylene lactones II, III and IV with aromatic thiols, azide anion, secondary amines and sodium enolates XI, XII and V was studied. The stereochemical outcome of this substitution is discussed from the viewpoint of mechanism of nucleophilic vinylic substitution (SNV).
Cathodic Reduction of Isatin-3-oxime
Authors: Hudák Alexander , Košturiak Adam , Hanudeľ Alexander , Meľuch Pavol
Published: April 2003

The molecule of isatin-3-oxime was studied polarografically over the region of pH 1 to 13. Cathodic reduction within the region of pH 1 to 10.5 gives rise to a single four-electron polarographic wave due to the reduction of the oxime group, whereas two polarographic waves are observed at pH 10.5 to 13: one is due to the reduction of the isatin-3-oxime anion, the other, more negative wave is associated with the reduction of the oxime group in the α-oxime isatinic acid anion, which is the product of hydrolytic cleavage of the isatin ring in the basic pH region. The experimental results give evidence that the substance can exist as seven ionic and molecular species in dependence on pH. The species have been confirmed polarographically, by IR and electronic spectroscopy, potentiometrically and by paper electrophoresis.
Determination of Composition of Reaction Mixtures from Alkylation of Toluene on Phosphorus-Modified H-ZSM-5 Zeolite
Authors: Mravec Dušan , Herain Jiří , Holotík Štefan
Published: April 2003

Determination of analytical composition of reaction mixtures formed by alkylation of toluene with ethylene on a phosphorus-modified H-ZSM-5 zeolite was made by a capillary high resolution gas chromatography. Identification of individual components of these reaction mixtures was performed by GS-MS method, using the samples obtained at 320 and 400 °C on H-ZSM-5 catalyst (modul 43.6) modified with 3.4 wt.% phosphorus at toluene to ethylene molar ratio 4.5 : 1 and the catalyst loading expressed as weight hour space velocity WHSV (for toluene) = 6.9 h-1. The reaction mixtures contained a total of 86 hydrocarbons. It was confirmed that in the addition to the main alkylation reaction, there proceeds also ethylene oligomerization with subsequent cyclization and aromatization, disproportionation, alkylation and cyclization reactions of alkylaromatic hydrocarbons to give alkylnaphthalenes and alkylindanes.
Kinetics of Catalytic Dehydration of 1-Pentanol
Authors: Vašutová Iveta , Králik Milan , Hronec Milan
Published: April 2003

Kinetic data of 1-pentanol dehydration on γ-alumina catalyst modified by potassium hydroxide were obtained using a continuous reactor with an internal recirculation. The conversion of 1-pentanol on this catalyst in the temperature range 300 - 390 °C and space velocity 1 - 8 kg (h kg)-1 (molar fraction of water in the feed was in the range 0 - 0.56) was 50 - 98% and the selectivity with respect to 1-pentene was 50 - 84%. The following six reactions have been taken into account to describe the catalytic dehydration of 1-pantanol: direct formation of 1-pentene from 1-pentanol, formation of bis(1-pentyl) ether from 1-pentanol, disproportionation of the ether to 1-pentanol and 1-pentene, formation of 1-pentene from the ether, isomerization of 1-pentene to 2-pentene and hydrolysis of the ether to 1-pentanol. Treatment of experimental data by Langmuir-Hinshelwood models showed that the model involving adsorption of 1-pentanol accompanied by dissociation is the most suitable one.
Quinazolinone Derivatives of Biological Interest V. Novel 4(3H)-Quinazolinones with Sedative-Hypnotic, Anticonvulsant and Antiinflammatory Activities
Authors: Abdel-Alim Abdel-Alim M. , El-Shorbagi Abdel-Nasser A. , El-Gendy Mahmoud A. , El-Shareif Hosny A. H.
Published: April 2003

2-Methyl-4(3H)-quinazolines carrying alkyl, cycloalkyl, aralkyl or aryl substituents at N-3 of the quinazoline ring exhibit analgetic, antipyretic and antiinflammatory activities comparable to those of aspirin and phenylbutazone. In our previous work, various 4(3H)-quinazoline derivatives were prepared. The present communication is a continuation of our effort in this field.
Heteropoly Acids Supported on α-Al2O3 as Solid Acid Catalysts for Methanol Transformation
Authors: Fikry Ebeid M. , Abdel-Ghaffar Ali , Anwar Amin , Aboul-Fotouh Sameh
Published: April 2003

Transformation of methanol into dimethyl ether and methane was carried out over two heteropoly acids supported on α-Al2O3 under atmospheric pressure and at 300 °C. The samples were characterized physicochemically (SBET, X-ray diffraction and acidity). The effect of reaction time, temperature, methanol partial pressure and of the content of the supported acid were studied. The results showed that α-Al2O3 increased the catalyst life. The selectivity towards dimethyl ether and methane formation depended on the type of acid and its content. The results indicate that dimethyl ether is formed by surface region whereas methane formation proceeds in the bulk of the catalyst.
Synthesis of Some Heterocyclic Sulfones Related to Quinolinol
Authors: Abdel Hafez Ali A.
Published: April 2003

It is well known that pyrazolone derivatives possess antifungal and antibacterial activities. Pyrazole and isoxazole derivatives are widely used in medicinal chemistry. In continuation of our work on the synthesis of heterocycles containing the quinoline moiety we synthesized different heterocyclic sulfones related to quinolinol.
Cloud Point Curves of Aqueous Solutions of Poly(N-ethylmethacrylamide)
Authors: Bohdanecký Miloslav , Horský Jiří , Petrus Vladimír , Mrkvičková Libuše , Ulbrich Karel
Published: April 2003

Aqueous solutions of poly(N-ethylmethacrylamide) have a lower critical solution temperature (LCST). Cloud point curves of five polydisperse samples (Mw . 10-6 = 0.06 to 2.04) in aqueous solutions at concentrations from 0.002 to 0.1 g/ml were obtained. The threshold concentration was found to be almost independent of the molecular weight. The threshold temperature decreases moderately with increasing M and the limiting value for infinite M obtained by the Shultz-Flory method is ΘLPE = 340.5 K. This value is lower by 2.5 K than the temperature ΘLη at which the intrinsic viscosity is proportional to the square root of the molecular weight. The difference cannot be assigned to experimental errors. The entropy-of-dilution parameter is negative (ψ = -2) and much higher than the value obtained from dilute solution properties. The differences in Θ and ψ values are discussed in terms of theory of polymer solutions where a higher-order interaction parameter is included.
Polystyrene in N-Methylpyrrolidone: Intrinsic Viscosity-Molar Weight Relations and Polystyrene Unperturbed Dimensions
Authors: Konáš Martin , Shultz Allan R.
Published: April 2003

Intrinsic viscosity-molar weight relations are reported for polystyrene in N-methylpyrrolidine (NMP) and NMP/LiBr solutions at 30, 45, 60, 75, 90 and 105 °C. Also, Mark-Houwink constants KΘ and the unperturbed dimensions of the polystyrene chains at these temperatures are calculated by applying Stockmayer-Fixman extrapolations of the viscosity/molar weight data. The use of in-line size exclusion chromatography and viscometry is shown to be a facile and accurate method for such studies.
Molecular Structure of a Cyclic Dimeric Model of Poly(6-hexanelactam)
Authors: Schneider Bohdan , Kvarda Jan , Dybal Jiří , Schmidt Pavel , Suchopárek Miloš , Prokopová Irena
Published: April 2003

Two crystalline forms of the cylindric dimer of 6-hexanelactam were studied by DSC, Raman, IR, NMR and computational methods. It was found that the high-temperature modification contains the energetically most favoured conformational structure, the low temperature modification contains the energetically less favoured conformation stabilized by stronger hydrogen bonds.
Block Copolymers Poly(2,6-dimethyl-1,4-phenylene oxide)-poly(6-hexanelactam). Physical Properties
Authors: Stehlíček Jaroslav , Kovářová Jana , Lednický František
Published: April 2003

In powdery poly(2,6-dimethyl-1,4-phenylene oxide)-poly(6-hexanelactam) diblock copolymers, the occurrence of an amorphous interphase consisting of both the blocks was indirectly proved by calorimetric and sorption methods. The copolymer shows a compatibilizing effect in solution-cast films made from the blends of both homocopolymers.
Quaternized Poly(2-dimethylaminoethyl methacrylate)-PVC Composite Membranes
Authors: Kůdela Vlastimil , Ševčík Stanislav , Ryba Olen , Vacík Jiří
Published: April 2003

A series of homogeneous and heterogeneous composite membranes consisting of PVC and crosslinked quaternized poly(2-dimethylaminoethyl methacrylate) was investigated electrochemically. Their electrolytic resistances ranged from nearly zero to insulator values at a permselectivity of 62 - 96.7%. Highly conductive and permselective membranes are promising for neutralization dialysis and dialysis of acids. Isolator-like membranes behave as bipolar ones (diode effect) and, in contact with KCl solutions of different concentrations. like electrets (persistent charge).
A Discussion of the Cyclotrimerization Mechanism of Isocyanates
Authors: Krakovský Ivan , Špírková Milena
Published: April 2003

A reaction scheme of cyclotrimerization of phenyl isocyanate in the presence of potassium 2-ethylhexanoate as a catalyst was proposed. From kinetic equations, the reaction orders and concentrations of the intermediates were calculated using quasistationary approximation. Concentrations of biuret and urea (product of reaction intermediates with water) can provide information on the reaction mechanism.
Crystal Structure and Conformation of Ring A of 2β-Bromo-19β,28-epoxy-18α-oleanan-3-one
Authors: Novotný Jiří , Podlaha Jaroslav , Klinot Jiří
Published: April 2003

The crystal structure of β-bromo-19β,28-epoxy-18α-oleanan-3-one was elucidated. The crystal is orthorhombic, P212121, a = 9.686(1), b = 14.355(2), c = 19.687(4) Å, Z = 4, R = 0.042 for 2 410 observed reflections. Rings B, C, D and E adopt the chair conformation, the five membered ether cycle in ring E occurs in the envelope form. Ring A takes the twist-boat conformation turned towards the classical boat with C2 and C5 in the stem-stern position, in contrast to the conformation in solution, which is turned in the opposite direction towards the classical boat with C3 and C10 in the stem-stern positions.
Age-adjusted International Prognostic Index predicts autologous stem cell transplantation outcome for patients with relapsed or primary refractory diffuse large B-cell lymphoma
Authors: Hamlin Paul A. , Zelenetz Andrew D. , Kewalramani Tarun , Qin Jing , Satagopan Jaya M. , Verbel David , Noy Ariela , Portlock Carol S. , Straus David J. , Yahalom Joachim , Nimer Stephen D. , Moskowitz Craig H.
Published: April 2003

Second-line chemotherapy followed by high-dose therapy (HDT) with autologous stem cell transplantation (ASCT) cures less than half of the patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). Prognostic models capable of predicting outcome are essential. In 3 sequential clinical trials, conducted from January 1993 to August 2000, we treated 150 patients with relapsed or primary refractory DLBCL with ifosfamide, carboplatin, and etoposide (ICE) chemotherapy followed by HDT/ASCT for patients with chemosensitive disease. We evaluated the age-adjusted International Prognostic Index at the initiation of second-line therapy (sAAIPI) as a predictor of progression-free survival (PFS) and overall survival (OS). At a median follow-up of 4 years, the PFS and OS are 28% and 34% by intention to treat and 39% and 45% for only those patients with chemosensitive disease. Three risk groups with different PFS and OS were identified by the sAAIPI: low risk (0 factors), 70% and 74%; intermediate risk (1 factor), 39% and 49%; and high risk (2 or 3 factors), 16% and 18% (P < .001 for both PFS and OS). The sAAIPI also predicts the PFS and OS for patients with ICEchemosensitive disease: low risk, 69% and 83%; intermediate risk, 46% and 55%; and high risk, 25% and 26% (P < .001 PFS and OS). The sAAIPI predicts outcome for patients with relapsed or primary refractory DLBCL in both intent-to-treat and chemosensitive populations. This powerful prognostic instrument should be used to evaluate new treatment approaches and to compare results of different regimens.
The Frobenius–Harper technique in a general recurrence model
Authors: Warren Di
Published: April 2003

We present a general recurrence model which provides a conceptual framework for well-known problems such as ascents, peaks, turning points, Bernstein's urn model, the Eggenberger–Pólya urn model and the hypergeometric distribution. Moreover, we show that the Frobenius-Harper technique, based on real roots of a generating function, can be applied to this general recurrence model (under simple conditions), and so a Berry–Esséen bound and local limit theorems can be found. This provides a simple and unified approach to asymptotic theory for diverse problems hitherto treated separately.
A probabilistic proof of Stein's factors
Authors: Xia Aihua
Published: April 2003

We provide a probabilistic proof of the Stein's factors based on properties of birth and death Markov chains, solving a tantalizing puzzle in using Markov chain knowledge to view the celebrated Stein–Chen method for Poisson approximations. This work complements the work of Barbour (1988) for the case of Poisson random variable approximation.
Elementary new proofs of classical limit theorems for Galton–Watson processes
Authors: Geiger Jochen
Published: April 2003

Classical results describe the asymptotic behaviour of a Galton–Watson branching process conditioned on non-extinction. We give new proofs of limit theorems in critical and subcritical cases. The proofs are based on the representation of conditioned Galton–Watson generation sizes as a sum of independent increments which is derived from the decomposition of the conditioned Galton–Watson family tree along the line of descent of the left-most particle.