[Influence of personal attitude of the manager on antibiotic use in pig production]. / Einfluss der persönlichen Einstellung des Betriebsleiters auf den Antibiotika-Verbrauch in der Schweineproduktion.

The attitude as well as the expertise of a person affect the behavior and actions in daily life. To investigate the influence of attitude and knowledge of pig producers on the use of antibiotics in farms, 220 Swiss pig producers were questioned on health awareness, attitude towards sustainable production, risk behavior, intrinsic motivation and knowledge about antibiotics and resistance development. In addition, the strategy of antibiotic use (therapeutic or prophylactic) and the business practice (single or group therapy) for the amount of antibiotics on one hand and for the risk of antibiotic resistance development on the other hand, were determined in a personal interview. Farmers using antibiotics only therapeutically had a better business practice. A direct link between the personal attitude and the antibiotic use or a higher risk of development of antibiotic resistance was not found in this investigation.

Anticodon nucleases.

A tRNALys-specific anticodon nuclease is kept in a latent form in a rare Escherichia coli strain, complexed with a DNA restriction enzyme. A phage T4 inhibitor of DNA restriction activates anticodon nuclease, but other T4 proteins restore tRNALys. Detection of a homologous system in Neisseria and a different anticodon nuclease in colicin E5 suggest ubiquity and diversity of such tRNA toxins. Analysis of these systems could reveal novel RNA recognition and cleavage mechanisms.

CD4+ CCR5+ T-cell dynamics during simian immunodeficiency virus infection of Chinese rhesus macaques.

Simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) provides a reliable model to study the relationship between lentivirus replication, cellular immune responses, and CD4+ T-cell dynamics. Here we investigated, using SIVmac251-infected RMs of a Chinese genetic background (which experience a slower disease progression than Indian RMs), the dynamics of CD4+ CCR5+ T cells, as this subset of memory/activated CD4+ T cells is both a preferential target of virus replication and a marker of immune activation. As expected, we observed that the number of circulating CD4+ CCR5+ T cells decreases transiently at the time of peak viremia. However, at 60 days postinfection, i.e., when set-point viremia is established, the level of CD4+ CCR5+ T cells was increased compared to the baseline level. Interestingly, this increase correlated with faster disease progression, higher plasma viremia, and early loss of CD4+ T-cell function, as measured by CD4+ T-cell count, the fraction of memory CD4+ T cells, and the recall response to purified protein derivative. Taken together, these data show a key difference between the dynamics of the CD4+ CCR5+ T-cell pool (and its relationship with disease progression) in Chinese RMs and those described in previous reports for Indian SIVmac251-infected RMs. As the SIV-associated changes in the CD4+ CCR5+ T-cell pool reflect the opposing forces of SIV replication (which reduces this cellular pool) and immune activation (which increases it), our data suggest that in SIV-infected Chinese RMs the impact of immune activation is more prominent than that of virus replication in determining the size of the pool of CD4+ CCR5+ T cells in the periphery. As progression of HIV infection in humans also is associated with a relative expansion of the level of CD4+ CCR5+ T cells, we propose that SIV infection of Chinese RMs is a very valuable and important animal model for understanding the pathogenesis of human immunodeficiency virus infection.

The middle subunit of replication protein A contacts growing RNA-DNA primers in replicating simian virus 40 chromosomes.

The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, its size distribution, its RNA primer content, and its replication fork polarity were determined [corrected]. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase alpha-primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging-strand DNA synthesis are evaluated in view of our results.

Cloning of nascent monkey DNA synthesized early in the cell cycle.

To study the structure and complexity of animal cell replication origins, we have isolated and cloned nascent DNA from the onset of S phase as follows: African green monkey kidney cells arrested in G1 phase were serum stimulated in the presence of the DNA replication inhibitor aphidicolin. After 18 h, the drug was removed, and DNA synthesis was allowed to proceed in vivo for 1 min. Nuclei were then prepared, and DNA synthesis was briefly continued in the presence of Hg-dCTP. The mercury-labeled nascent DNA was purified in double-stranded form by extrusion (M. Zannis-Hadjopoulos, M. Perisco, and R. G. Martin, Cell 27:155-163, 1981) followed by sulfhydryl-agarose affinity chromatography. Purified nascent DNA (ca. 500 to 2,000 base pairs) was treated with mung bean nuclease to remove single-stranded ends and inserted into the NruI site of plasmid pBR322. The cloned fragments were examined for their time of replication by hybridization to cellular DNA fractions synthesized at various intervals of the S phase. Among five clones examined, four hybridized preferentially with early replicating fractions.

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Antiretroviral therapy of HIV-1 infection: established treatment strategies and new therapeutic options.

Recently, studies have shown that non-nucleoside reverse transcriptase inhibitors, such as efavirenz or nevirapine, in combination with two nucleoside analogues have an antiretroviral potency comparable to protease inhibitor containing regimens. Triple combination therapy that includes a non-nucleoside reverse transcriptase inhibitor can therefore be regarded as an effective alternative first-line treatment of HIV-1 infection.

Replication protein A modulates its interface with the primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes.

The eukaryal single-stranded DNA binding protein replication protein A (RPA) binds short oligonucleotides with high affinity but exhibits low cooperativity in binding longer templates, opposite to prokaryal counterparts. This discrepancy could reflect the smaller size of the replicative template portion availed to RPA. According to current models, this portion accommodates an RNA-DNA primer (RDP) of <40 nt (nested discontinuity) or a several-fold longer Okazaki fragment (initiation zone). Previous in situ UV-crosslinking revealed that RPA also interacts with nascent DNA, especially growing RDPs. Here we compare nascent SV40 DNA chains UV-crosslinked to the middle and large RPA subunits and use the data to re-examine the two models. The middle subunit interacted with the nascent chains after a few DNA residues were added to the RNA primer while the large subunit became accessible after extension by several more. Upon RDP maturation, the middle subunit disengaged while the large subunit remained accessible during further limited extension. A corresponding shift in preference in favor of the large subunit has been reported for purified RPA and synthetic gapped duplexes upon reduction of the gap from 19 to 9 nt. Combined, these facts support the proposal that the mature RDP faces downstream a correspondingly small gap, possibly created by removal of the RNA primer moiety from an adjacent, previously synthesized RDP (nested discontinuity) but insufficient for continuous elongation of the RDP into an Okazaki fragment (initiation zone).

Specific interaction between anticodon nuclease and the tRNA(Lys) wobble base.

The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease cleaves its natural substrate 5' to the wobble base, yielding 2',3'-cyclic phosphate termini. Previous work has implicated the anticodon of tRNA(Lys) as a specificity element and a cluster of amino acid residues at the carboxy-proximal half of PrrC in its recognition. We further examined these assumptions by assaying unmodified and hypomodified derivatives of tRNA(Lys) as substrates of wild-type and mutant alleles of PrrC. The data show, first, that the anticodon sequence and wobble base modifications of tRNA(Lys) play major roles in the interaction with anticodon nuclease. Secondly, a specific contact between the substrate recognition site of PrrC and the tRNA(Lys) wobble base is revealed by PrrC missense mutations that suppress the inhibitory effects of wobble base modification mutations. Thirdly, the data distinguish between the anticodon recognition mechanisms of PrrC and lysyl-tRNA synthetase.

Phage T4-coded Stp: double-edged effector of coupled DNA and tRNA-restriction systems.

The optional Escherichia coli prr locus encodes two physically associated restriction systems: the type IC DNA restriction-modification enzyme EcoprrI and the tRNA(Lys)-specific anticodon nuclease, specified by the PrrC polypeptide. Anticodon nuclease is kept latent as a result of this interaction. The activation of anticodon nuclease, upon infection by phage T4, may cause depletion of tRNA(Lys) and, consequently, abolition of T4 protein synthesis. However, this effect is counteracted by the repair of tRNA(Lys) in consecutive reactions catalysed by the phage enzymes polynucleotide kinase and RNA ligase. Stp, a short polypeptide encoded by phage T4, has been implicated with activation of the anticodon nuclease. Here we confirm this notion and also demonstrate a second function of Stp: inhibition of EcoprrI restriction. Both effects depend, in general, on the same residues within the N-proximal 18 residue region of Stp. We propose that Stp alters the conformation of EcoprrI and, consequently, of PrrC, allowing activation of the latent anticodon nuclease. Presumably, Stp evolved to offset a DNA restriction system of the host cell but was turned, eventually, against the phage as an activator of the appended tRNA restriction enzyme.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Detection of anticodon nuclease residues involved in tRNALys cleavage specificity.

The tRNALys-specific anticodon nuclease exists in latent form in Escherichia coli strains containing the optional prr locus. The latency is a result of a masking interaction between the anticodon nuclease core-polypeptide PrrC and the Type IC DNA restriction-modification enzyme EcoprrI. Activation of the latent enzyme by phage T4-infection elicits cleavage of tRNALys 5' to the wobble base, yielding 5'-OH and 2', 3'-cyclic phosphate termini. The N-proximal half of PrrC has been implicated with (A/G) TPase and EcoprrI interfacing activities. Therefore, residues involved in recognition and cleavage of tRNALys were searched for at the C-half. Random mutagenesis of the low-G+C portion encoding PrrC residues 200-313 was performed, followed by selection for loss of anticodon nuclease-dependent lethality and production of full-sized PrrC-like protein. This process yielded a cluster of missense mutations mapping to a region highly conserved between PrrC and two putative Neisseria meningitidis MC58 homologues. This cluster included two adjacent members that relaxed the inherent enzyme's cleavage specificity. We also describe another mode of relaxed specificity, due to mere overexpression of PrrC. This mode was shared by wild-type PrrC and the other mutant alleles. The additional substrates recognised under the promiscuous conditions had, in general, anticodons resembling that of tRNALys. Taken together, the data suggest that the anticodon of tRNALys harbours anticodon nuclease identity elements and implicates a conserved region in PrrC in their recognition.

Nucleotide and deduced amino acid sequence of stp: the bacteriophage T4 anticodon nuclease gene.

Pre-existing host tRNAs are reprocessed during bacteriophage T4 infection of certain Escherichia coli strains. In this pathway, tRNALys is cleaved 5' to the wobble base by anticodon nuclease and is later restored in polynucleotide kinase and RNA ligase reactions. Anticodon nuclease depends on prr, a locus found only in host strains that restrict T4 mutants lacking polynucleotide kinase and RNA ligase; and on stp, the T4 suppressor of prr restriction. stp was cloned and the nucleotide sequences of its wild-type and mutant alleles determined. Their comparison defined an stp open reading frame of 29 codons at 162.8 to 9 kb of T4 DNA (1 kb = 10(3) base-pairs). We suggest that stp encodes a subunit of anticodon nuclease, perhaps one that harbors the catalytic site; while additional subunits, such as a putative prr gene product, impart protein folding environment and tRNA substrate recognition.

Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X.

Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli). The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions. Inactivating mutations in stp suppress pnk- or rli- mutations in E. coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity. We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp. It has been shown that a single locus in E. coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants. A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity. However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.

[Salt, hypertension and cardiovascular risk]. / Salz, Hypertonie und kardiovaskuläres Risiko.

For more than a century salt and blood pressure have been linked. Prospective randomized clinical trials and meta-analyses provided evidence that a reduction of daily salt intake of 100 mmol will lower systolic blood pressure by 3-5 mm Hg, whereas diastolic blood pressure can be reduced by 1 mm Hg. The effect of salt restriction depends strongly on individual salt sensitivity. Whether a reduction of salt intake will ultimately result in a decline of cardiovascular morbidity and mortality remains to be determined in future studies.

Long-term immunological response in HIV-1-infected subjects receiving potent antiretroviral therapy.

OBJECTIVE: To determine the long-term T-lymphocyte response to highly active antiretroviral therapy (HAART) and to define predictors of the immunological response. DESIGN: Cohort study, including 135 HIV-1-infected subjects at a city general practice who commenced HAART between 1996 and 1998. METHODS: Collection of plasma HIV-1 RNA, CD4+ and CD8+ T-lymphocyte data at 3-6 monthly time intervals over 2 years. RESULTS: Seventy-three subjects (54%) achieved suppression of plasma HIV-1 RNA to levels below 400 copies/ml during the observation period, 31 individuals (23%) had detectable plasma HIV-1 RNA below 10,000 copies/ml and 31 subjects (23%) had virological failures with viral loads above 10,000 copies/mL. Median CD4+ T lymphocytes increased from 246 to 463 x 10(6) cells/l, showing a median rise of 20 x 10(6) cells/l per month in the first 3 months and 7 x 10(6) cells/l per month thereafter. The proportion of individuals who reached CD4+ cell counts above 500 x 10(6) cells/l increased from 8% at baseline to 54% at 2 years. Treatment-naïve individuals, subjects with a large reduction of HIV-1 RNA or a large early CD8+ increase had better early CD4+ responses. Long-term CD4+ T-cell increases were inversely correlated with mean plasma HIV-1 RNA levels. Baseline CD4+ T-cell count was the most important determinant of reaching CD4+ cell counts above 500 x 10(6) cells/l. Nineteen per cent of subjects had no further CD4+ T-cell increases in the second year of therapy despite undetectable viral load. CONCLUSIONS: Immune reconstitution is a slow process, showing a large individual variability. The virological response to HAART was the most important determinant of the immunological short- and long-term response.

Rapid restoration of CD4 T cell subsets in subjects receiving antiretroviral therapy during primary HIV-1 infection.

OBJECTIVE: To compare the effect of highly active antiretroviral therapy on immune reconstitution in subjects with acute and chronic HIV-1 infection. DESIGN: Prospective study including 58 treatment-naive subjects who commenced indinavir or nelfinavir and two nucleosides during primary (PHI; n = 28) or chronic HIV-1 infection (CHI; n = 30). METHODS: Naive (CD45RA+ 62L+), memory (CD45RA-) and activated (CD38+ HLA-DR+) T cell subsets were quantified at 1-2 monthly time intervals using 4-colour flow cytometry. RESULTS: At 1 year, HIV-1 RNA declined in both cohorts to undetectable levels (< 50 copies/ml), while median CD4 lymphocyte count increased from 470 to 758 x 10(6) cells/l in PHI and from 204 to 310 x 10(6) cells/l in CHI, reaching > 500 x 10(6) cells/l in 93% of PHI, but only in 37% of CHI subjects (P < 0.001). Naive CD4 lymphocytes increased from 106 to 176 x 10(6) cells/l in PHI and from 41 to 44 x 10(6) cells/l in CHI (PHI versus CHI at 12 months: P = 0.003), while memory cells rose from 368 to 573 x 10(6) cells/l in PHI and from 148 to 223 x 10(6) cells/l in CHI (P < 0.001). Early increases (< 3 months) of CD4 lymphocytes were larger in subjects with PHI, consisting of naive CD45RA+ CD62L+ as well as memory CD45RA- CD62L+ cells (P = 0.001). CD4 activation declined from 5 to 2% in PHI and from 13 to 6% in CHI (P = 0.001), while CD8 cell activation was reduced from 33 to 15% in PHI and from 42 to 19% in CHI (P = 0.02). CONCLUSION: Immune reconstitution was more complete, occurred earlier and comprised both naive and memory CD4 T lymphocytes in subjects who commenced antiretroviral therapy during primary HIV-1 infection.

Treatment response and durability of a double protease inhibitor therapy with saquinavir and ritonavir in an observational cohort of HIV-1-infected individuals.

OBJECTIVE: To evaluate treatment response, durability and tolerance of a four-drug regimen including saquinavir and ritonavir in combination with either zidovudine/lamivudine or stavudine/lamivudine. DESIGN: Observational cohort of HIV-positive individuals. METHODS: Viral load, CD4+ and CD8+ T lymphocyte counts were assessed at intervals of 1-3 months in subjects commencing therapy between July 1996 and November 1996. Adverse events were evaluated as well as risk factors for therapeutic failures. RESULTS: A group of 56 male patients were included and followed for 48 weeks. Of these, 66% had already taken a protease inhibitor. Viral load dropped by a median 1.98 log10 HIV RNA copies/ml from baseline (interquartile range: 1.49-2.46) and became undetectable (< 400 copies/ml) in 68% of patients. Response varied: 9% were non-responders (HIV RNA reduction < 0.5 log10 copies/ml) and 23% were incomplete responders (nadir of HIV RNA > 400 copies/ml). After 48 weeks, viral load remained undetectable in 49%. Median CD4+ T lymphocyte count increased from 191 x 10(6) to 418 x 10(6) cells/l (range, 241-537 x 10(6) cells/l). Although protease inhibitor and nucleoside pretreatment selected for drug-resistant viral mutants, only the protease inhibitor experience was identified as a risk factor for therapeutic failure. Adverse events occurred in 73% of patients and led to a change of therapy in 9%. CONCLUSION: Despite advanced HIV disease and pretreatment with multiple antiretroviral drugs, a strong initial treatment response to this drug regimen was observed. However, virological failure occurred in 51% of patients after 48 weeks and frequent adverse events complicated therapy.

N-acetyltransferase 2 polymorphism in patients infected with human immunodeficiency virus.

OBJECTIVES: To evaluate the prevalence of slow acetylation of hepatic N-acetyltransferase 2 (NAT2) in patients with different stages of human immunodeficiency virus (HIV) infection, to assess the relationship between acetylation capacity and the degree of immunosuppression, and to study the concordance between NAT2 phenotype and genotype. METHODS: This prospective study in a consecutive sample of HIV-infected patients was performed in the outpatient department of a university hospital that provides primary and tertiary care. The NAT2 genotype was assessed by polymerase chain reaction and restriction fragment length polymorphism, the NAT2 phenotype was determined by caffeine test (urinary metabolic ratio of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine). RESULTS: Fifty patients with Centers for Disease Control HIV infection stages A (10 patients), B (20 patients), and C (20 patients) were included in the study after each gave informed consent. According to genotyping and phenotyping, 32 (64%) patients were slow acetylators, with a concordance of the two methods of 96%. The overall distribution was similar to distributions reported in other white populations. The slow acetylator phenotype was found in seven, 16, and nine patients with stage A, B, and C, respectively. Eight of the 10 patients with previous adverse reactions to sulfonamides had slow acetylator phenotypes. Acetylation capacity was independent of CD4 cell counts. CONCLUSIONS: This study revealed an excellent agreement between genotypes and phenotypes of NAT2 in patients with HIV infection. There was no increase in prevalence of slow acetylation in patients with advanced stages of the disease. This apparent discrepancy to an earlier study may be the result of differences in co-medication of the patients studied and may point to the relevance of drug interactions in the treatment of patients with HIV infection.

Pharmacodynamics of 3-hydroxyquinidine alone and in combination with quinidine in healthy persons.

The relation between serum concentration of 3-hydroxyquinidine (3-OHQ), a major metabolite of quinidine in humans, and the pharmacologic effect alone and in combination with the parent drug was studied. The heart rate-corrected, computer-averaged QT interval (QTc) was used as the pharmacologic endpoint. In a randomized, double-blind study, 5 healthy subjects received, on 3 separate days 1 week apart, either (1) 300 to 400 mg 3-OHQ orally or (2) 150 mg quinidine base intravenously or (3) a combination in the same doses. Blood samples and electrocardiographic recordings were obtained over the following 10 hours. Serum concentrations of 3-OHQ and quinidine were determined by high-pressure liquid chromatography and the free fraction by ultrafiltration. Peak concentrations of 3-OHQ varied between 1,362 and 3,480 ng/ml after oral 3-OHQ ingestion, but were negligible after intravenous quinidine infusion. The free fraction was 49% +/- 4.8 (mean +/- standard deviation) for 3-OHQ and 20% +/- 4.3 for quinidine. In all 5 subjects a statistically significant correlation was found between serum concentration and QTc prolongation for both quinidine and 3-OHQ (largest p value less than 0.025). The mean slope of the regression line was 0.0184 +/- 0.0128 for 3-OHQ and 0.0297 +/- 0.0111 for quinidine. Multiple linear regression revealed in each subject a significant additive effect of 3-OHQ when administered together with quinidine (largest p value less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)