Analysis of a pleiotropic gene region involved in formation of catalytically active hydrogenases in Alcaligenes eutrophus H16

In Alcaligenes eutrophus H16 a pleiotropic DNA-region is involved in formation of catalytically active hydrogenases. This region lies within the hydrogenase gene cluster of megaplasmid pHG1. Nucleotide sequence determination revealed five open reading frames with significant amino acid homology to the products of the hyp operon of Escherichia coli and other hydrogenase-related gene products of diverse organisms. Mutants of A. eutrophus H16 carrying Tn5 insertions in two genes (hypB and hypD) lacked catalytic activity of both soluble (SH) and membrane-bound (MBH) hydrogenase. Immunological analysis showed that the mutants contained SH-and MBH-specific antigen. Growing the cells in the presence of ⁶³Ni²⁺ yielded significantly lower nickel accumulation rates of the mutant strains compared to the wild-type. Analysis of partially purified SH showed only traces of nickel in the mutant protein suggesting that the gene products of the pleiotropic region are involved in the supply and/or incorporation of nickel into the two hydrogenases of A. eutrophus.     

Two-and three-dimensional HCN experiments for correlating base and sugar resonances in 15N, 13C-labeled RNA oligonucleotides

Summary New 2D and 3D ¹H-¹³C-¹⁵N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in ¹³C-and ¹⁵N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective ¹³C-refocusing and ¹⁵N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D ¹H-¹⁵N and 3D¹H-¹³C-¹⁵N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV.     

Influence of Different Intensities of Pesticide Management on the Structure of Ground Beetle Communities (Coleoptera; Carabidae) During a Crop Rotation

Investigations were lead through in the years 1992 to 1995 at Halle (Saale) on a stand 24ha in size. Aim of the survey was to record the effects of graduate intensities of pest management on ground beetles. For this purpose 6 plots were installed on the field, 72 ‐ 200m each. Two plots served as control areas, without any application of pesticides. On two other plots intensive chemical pest management was applied. On the last two plots treatments were lead through considering the economic thresholds for weeds, fungal and insect pests according to the rules of integrated pest management. Ground beetles were sampled by using pitfall traps. In 1992 complementary methods to the pitfall trap catches, pick up of beetles after pesticide applications and semi-field-tests, on all plots were lead through. The graduate pest management was carried out 1992 and 1993 in winter wheat and 1994 in sugar beet stands. The last crop in 1995 was summer barley. In this year all plots were treated conventionally. In 1992 the differences between the total catches of ground beetles on the experimental sites were not high. In the following year most species were predominant on the control sites. In 1994 in sugar beet mechanical weed control was carried out on the control plots. The intensity of pesticide management between intensive and integrated plots differs only small. The amounts of ground beetles reached similar values on all experimental sites. In contrast to the expections the trap catches of ground beetles in the final investigations in summer barley were highest in the plots which had been treated integrated in the years before. The smallest amount was sampled in the former intensively managed sites. Renunciation of pesticides often is connected with economic losses which are not tolerable. If management is conducted considering the economic thresholds corresponding to the principles of integrated pest management negative effects on the economy of nature should not be expected for the longer term.     

Versatile retargeting of SH3 domain binding by modification of non-conserved loop residues

Src-homology (SH3) domain belongs to a class of ubiquitous modular protein domains found in nature. SH3 domains have a conserved surface that recognises proline-rich peptides in ligand proteins, but additional contacts also contribute to binding. Using the SH3 domain of hematopoietic cell kinase as a test case, we show that SH3 binding properties can be profoundly altered by modifications within a hexapeptide sequence in the RT-loop region that is not involved in recognition of currently known consensus SH3 target peptides. These results highlight the role of non-conserved regions in SH3 target selection, and introduce a strategy that may be generally feasible for generating artificial SH3 domains with desired ligand binding properties.     

HIV-1 RNA Levels and Antiretroviral Drug Resistance in Blood and Non-Blood Compartments from HIV-1–Infected Men and Women enrolled in AIDS Clinical Trials Group Study A5077

Background

Detectable HIV-1 in body compartments can lead to transmission and antiretroviral resistance. Although sex differences in viral shedding have been demonstrated, mechanisms and magnitude are unclear. We compared RNA levels in blood, genital-secretions and saliva; and drug resistance in plasma and genital-secretions of men and women starting/changing antiretroviral therapy (ART) in the AIDS Clinical Trials Group (ACTG) 5077 study.

Methods

Blood, saliva and genital-secretions (compartment fluids) were collected from HIV-infected adults (≥13 years) at 14 United-States sites, who were initiating or changing ART with plasma viral load (VL) ≥2,000 copies/mL. VL testing was performed on all compartment fluids and HIV resistance genotyping on plasma and genital-secretions. Spearman rank correlations were used to evaluate concordance and Fisher’s and McNemar’s exact tests to compare VL between sexes and among compartments.

Results

Samples were available for 143 subjects; 36% treated (23 men, 29 women) and 64% ‘untreated’ (40 men, 51 women). RNA detection was significantly more frequent in plasma (100%) than genital-secretions (57%) and saliva (64%) (P<0.001). A higher proportion of men had genital shedding versus women (78% versus 41%), and RNA detection was more frequent in saliva versus genital-secretions in women when adjusted for censoring at the limit of assay detection. Inter-compartment fluid VL concordance was low in both sexes. In 22 (13 men, 9 women) paired plasma-genital-secretion genotypes from treated subjects, most had detectable resistance in both plasma (77%) and genital-secretions (68%). Resistance discordance was observed between compartments in 14% of subjects.

Conclusions

HIV shedding and drug resistance detection prior to initiation/change of ART in ACTG 5077 subjects differed among tissues and between sexes, making the gold standard blood-plasma compartment assessment not fully representative of HIV at other tissue sites. Mechanisms of potential sex-dependent tissue compartmentalization should be further characterized to aid in optimizing treatment and prevention of HIV transmission.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00007488","term_id":"NCT00007488"}}NCT00007488     

Biomarkers of Plasmodium falciparum Infection during Pregnancy in Women Living in Northeastern Tanzania

In pregnant women, Plasmodium falciparum infections are an important cause of maternal morbidity as well as fetal and neonatal mortality. Erythrocytes infected by these malaria-causing parasites accumulate through adhesive interactions in placental intervillous spaces, thus evading detection in peripheral blood smears. Sequestered infected erythrocytes induce inflammation, offering the possibility of detecting inflammatory mediators in peripheral blood that could act as biomarkers of placental infection. In a longitudinal, prospective study in Tanzania, we quantified a range of different cytokines, chemokines and angiogenic factors in peripheral plasma samples, taken on multiple sequential occasions during pregnancy up to and including delivery, from P. falciparum-infected women and matched uninfected controls. The results show that during healthy, uninfected pregnancies the levels of most of the panel of molecules we measured were largely unchanged except at delivery. In women with P. falciparum, however, both comparative and longitudinal assessments consistently showed that the levels of IL-10 and IP-10 increased significantly whilst that of RANTES decreased significantly, regardless of gestational age at the time the infection was detected. ROC curve analysis indicated that a combination of increased IL-10 and IP-10 levels and decreased RANTES levels might be predictive of P. falciparum infections. In conclusion, our data suggest that host biomarkers in peripheral blood may represent useful diagnostic markers of P. falciparum infection during pregnancy, but placental histology results would need to be included to verify these findings.     

Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro.

Autologous cell mixtures containing T cells, B cells, and adherent accessory cells from individuals primed to the malaria parasite Plasmodium falciparum by repeated natural infections were investigated for induction of Ig and antibody secretion in vitro. In vitro activation of cell cultures with two synthetic peptides corresponding to immunodominant T cell epitopes of the merozoite Ag ring-infected erythrocyte surface Ag (Mr 155,000) (Pf155/RESA), one from its carboxyl-terminal repeat and one from its nonrepeated amino-terminal region, gave rise to significant IgG secretion. Supernatants from lymphocyte cultures activated with either one of these peptides contained antibodies reacting with P. falciparum Ag in immunofluorescence assays and with Pf155/RESA peptides in a slot blot assay. No anti-P. falciparum antibodies were induced in the medium controls by lymphocyte stimulation with either tetanus toxoid or PWM. Induction in vitro of anti-Pf155/RESA antibodies was correlated with the presence of such antibodies in the sera of the lymphocyte donors, suggesting that the induction of antibody secretion reflected a secondary response in vitro of in vivo primed cells. Inspection of antibody profiles in individual donors revealed that the peptide corresponding to a sequence in the 3' repeat region induced anti-Pf155/RESA peptide antibodies reacting with identical or related and cross-reacting sequences in the 3' or 5' repeat region of the molecule. In contrast, the peptide corresponding to a nonrepeated T cell epitope in the amino terminus of the molecule only induced antibodies to an immunodominant amino-terminal B cell epitope partly overlapping with the T cell reactive sequence. Similar findings were made in the lymphocyte donors' plasma, frequently displaying significant correlations between antibody reactivities to the repeat peptides but not between these reactivities and those to the amino-terminal peptide. The marked specificity of this antibody formation in vitro suggests an underlying process of cognate recognition involving Ag-specific T and B cells reacting with different segments of the inducer peptide. The present experimental system should be well suited for identification of Th epitopes capable of inducing the production of antibodies of defined specificity in the human system.     

T and B cell responses of Plasmodium falciparum malaria-immune individuals to synthetic peptides corresponding to sequences in different regions of the P. falciparum antigen Pf155/RESA

The C-terminal (3') amino acid repeat region of the Plasmodium falciparum Ag Pf155/RESA, a vaccine candidate, contains immunodominant T and B cell epitopes. In order to identify additional T cell epitopes in the molecule, synthetic peptides corresponding to the centrally (5') located repeat region, as well as to four nonrepeated regions, were synthesized. T cells from 46 P. falciparum-primed individuals living in a holoendemic area of The Gambia where malaria transmission is seasonal were tested for their responsiveness to the peptides by thymidine incorporation and IFN-gamma release. There was a considerable variation in the response to different peptides. Proliferation and IFN-gamma release were not correlated in individual donors, underlining the importance of measuring both activities when screening donor populations for total T cell responsiveness to a given Ag. Whereas 72% of the donors responded with proliferation and/or IFN-gamma release to the intact protein the mean % responders to the peptides was 40%. The most frequent responses (up to 60%) were induced with peptides from the 3'- and 5'-repeat region of the protein. Analysis of some closely related sequences in the 3'-repeat region indicated that they contained at least two epitopes that were either distinct or cross-reacting in different donors, suggesting difference in the genetic control of these responses. When the same peptides were investigated for reactivity with antibodies, the best T cell inducing sequences also displayed the best antibody reactivities. However, in individual donors, T and B cell responses were not correlated. T cell responses were shown to persist after a period with no P. falciparum transmission, whereas antibody concentrations tended to decrease, suggesting differences in the requirements of boosting at the T and B cell levels, respectively.     

Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion

Summary In dipteran insects the most distal telomere-associated DNA known to exist consists of long, complex tandem repeats. We have classified the 340-bp tandemly arranged repeats in Chironomus pallidivittatus. The repeats are distributed in a small number of subfamilies. One type of the repeat has the character of a master unit from which other main units can be derived usually by simple changes. The derived subfamilies contain segments that are degenerate versions of the corresponding segment in the master sequence. Such segments can also occur together in one and the same repeat unit in different combinations. There is a complete absence of subfamily-specific base variants in regions lying outside of the degenerate segments. Homogenization takes place between DNA sequences that are often smaller than a whole repeat unit. The mosaic structure of the repeat arrays suggests that gene conversion is an important force in the generation and maintenance of this family of repeats.Offprint requests to: M. Cohn