1H NMR conformational study on n-terminal nonapeptide sequences of HIV-1 Tat protein: a contribution to structure–activity relationships

On the basis of our recent results, the N-terminal sequence of HIV-1 Tat protein as a natural competitive inhibitor of dipeptidyl peptidase IV (DP IV) is supposed to interact directly with the active site of DP IV hence mediating its immunosuppressive effects via specific DP IV interactions. Of special interest is the finding that amino acid substitutions of the Tat(1–9) peptide (MDPVDPNIE) in position 5 with S-isoleucine and in position 6 with S-leucine led to peptides with strongly reduced inhibitory activity suggesting differences in the solution conformation of the three analogues. Therefore, ¹H NMR techniques in conjunction with molecular modelling have been used here to determine the solution structure of Tat(1–9), I⁵-Tat(1–9) and L⁶-Tat(1–9) and to examine the influence of amino acid exchanges on structural features of these peptides. The defined structures revealed differences in the conformations what might be the reason for different interactions of these Tat(1–9) analogues with certain amino acids of the active site of DP IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24

Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S₄ to S₄′) are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.     

Properties of ternary phospholipid/dimethyl sulfoxide/water systems at low temperatures

X-ray diffraction, neutron diffraction and differential scanning calorimetry were used to investigate phase transitions in the ternary system phospholipid/dimethyl sulfoxide (DMSO)/water under cooling for three homologous phospholipids: dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC). Below the temperature of ice formation from -40 to -113 degrees C, a new lamellar phase of DPPC and DSPC was found at and above a DMSO molar fraction of X(DMSO) = 0.05. Below X(DMSO) = 0.05 only a single dehydrated Lc-phase exists after ice formation. The new phase has an increased membrane repeat distance and coexists with a dehydrated Lc-phase. DPPC with a DMSO molar fraction of X(DMSO) = 0.07 shows a membrane repeat distance of the new phase of d = 6.61 +/- 0.03 nm. The value of d increases at the increase of X(DMSO). The new phase was not observed in the ternary system with DMPC. No correlation between the new phase and the glass transition of bound water in the intermembrane space was detected. The new phase was detected only in the systems with excess of water. The creation of the new phase demonstrates the specific DMSO interaction with hydrocarbon chains.     

Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system: 3. Plasma immunoglobulins and cytokines of workers with quantified moderately-increased body burdens

The concentrations of immunoglobulins (IgA, IgD, IgG, IgM) and of several cytokines were measured in the plasma of volunteers with clearly, but moderately, increased body burdens of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF), using monoclonal antibodies and an enzyme-linked immuno-sorbant assay. Two groups of workers with different body burdens of PCDD/PCDF were studied: (trial I) persons with mainly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and (trial II) persons with mainly penta- and hexachlorinated dibenzofurans (P5CDF/H6CDF) in their blood fat. Including the reference group, 158 volunteers were investigated. A slight but statistically significant decrease was observed in the plasma concentration of IgG1 in persons exposed to TCDD, but not in persons exposed to P5CDF/H6CDF. When the data of both groups were pooled and a multi-regression analysis against international TCDD toxicity equivalencies (I-TEq, NATO/CCMS) was performed, taking several confounding factors into account, no influence of the dioxin exposure could be revealed. There were no changes in the plasma concentrations of the other immunoglobulins studied. In the same volunteers, no deviation from the reference range was found for the concentrations of the cytokines: IL-1alpha, IL-1beta, IL-6 and TNFalpha in blood plasma.     

Novel 3-amino-2-hydroxy acids containing protease inhibitors. Part 1: Synthesis and kinetic characterization as aminopeptidase P inhibitors

Novel, potent inhibitors of aminopeptidase P, containing a 3-amino-2-hydroxy acid and a proline or a proline analogues, have been prepared. One part of the bestatin-derived inhibitors was found to inhibit APP from Escherichia coli and from rat intestine according to a mixed-type mechanism, with Ki values up to 1.26 microM. The other compounds, 3-amino-2-hydroxy acyl prolines of a different configuration, inhibit APP competitively, according to a slow-binding mechanism, with Ki values in the nanomolar up to the micromolar range.     

Identification and verification of novel rodent postsynaptic density proteins

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.     

Measurements of urinary leptin and capillary leptin: alternative tools for the assessment of the leptin status?

OBJECTIVES: The aim of this study was to check whether leptin is reliably measurable in urine samples of children, adolescents, and adults and to examine whether capillary leptin measurements can be utilized as an alternative tool to assess the leptin status. METHODS: Two studies were performed. In both studies, leptin was quantified by an ultrasensitive and highly specific enzyme immunoassay (ELISA; R & D Systems). Anthropometric measures were taken from all study subjects, and body fat was calculated using skinfold thickness measurements. In study 1, leptin was analyzed in 24-hour urine samples of 155 healthy children and adolescents and 26 healthy adults after a methodological modification of the assay necessary for urine analysis. In study 2, venous and capillary blood samples were collected in 26 healthy adults within 10 min on the same day. RESULTS: After adapting the assay system to urine matrix, the detection range was 20-160 pg/ml. Only in 2 of 181 urine samples reproducibly measurable urinary leptin concentrations in the lowest detection range were found. In study 2, a close correlation was found between log capillary and log venous leptin concentrations (r = 0.98, p < 0.001) and between log capillary as well as log venous leptin levels and percent body fat (r = 0.86, p < 0.001). CONCLUSIONS: Our results based on one of the most specific and sensitive ELISAs currently available show that leptin is generally undetectable in the urine from healthy children, adolescents and adults. Thus, urinary leptin excretion cannot be used as a noninvasive marker of the leptin status. Our findings in healthy adults show that the merely moderately invasive determination of capillary leptin allows a reliable assessment of the individual leptin status and may be used instead of venous leptin as a biochemical indicator of body fatness.     

Comparison of Commercially Available Target Enrichment Methods for Next-Generation Sequencing

Isolating high-priority segments of genomes greatly enhances the efficiency of next-generation sequencing (NGS) by allowing researchers to focus on their regions of interest. For the 2010–11 DNA Sequencing Research Group (DSRG) study, we compared outcomes from two leading companies, Agilent Technologies (Santa Clara, CA, USA) and Roche NimbleGen (Madison, WI, USA), which offer custom-targeted genomic enrichment methods. Both companies were provided with the same genomic sample and challenged to capture identical genomic locations for DNA NGS. The target region totaled 3.5 Mb and included 31 individual genes and a 2-Mb contiguous interval. Each company was asked to design its best assay, perform the capture in replicates, and return the captured material to the DSRG-participating laboratories. Sequencing was performed in two different laboratories on Genome Analyzer IIx systems (Illumina, San Diego, CA, USA). Sequencing data were analyzed for sensitivity, specificity, and coverage of the desired regions. The success of the enrichment was highly dependent on the design of the capture probes. Overall, coverage variability was higher for the Agilent samples. As variant discovery is the ultimate goal for a typical targeted sequencing project, we compared samples for their ability to sequence single-nucleotide polymorphisms (SNPs) as a test of the ability to capture both chromosomes from the sample. In the targeted regions, we detected 2546 SNPs with the NimbleGen samples and 2071 with Agilent''s. When limited to the regions that both companies included as baits, the number of SNPs was ∼1000 for each, with Agilent and NimbleGen finding a small number of unique SNPs not found by the other.     

Polarographische Bestimmung von Chlorat-Rückständen im Boden und in der Pflanze

An increase in the inoculum level of root‐knot nematode, Meloidogyne incognita and the reniform nematode, Rotylenchulus reniformis resulted in a relative decrease in plant growth parameters of chickpea. Consequently water absorption capability of roots was impaired. M. incognita caused greater reduction than R. reniformis at the same inoculum level. In concomitant inoculation of M. incognita and R. reniformis there was greater suppression in plant growth of chickpea. The suppression in concomitant inoculations was less than the sum of the suppression caused by the same levels of inoculations of the individual species. The multiplication rate of the nematodes decreased as the inoculum level increased. The results also suggest competition for feeding sites between the two nematode species. The multiplication rate of one species progressively decrease with the increase in the inoculum levels of the other nematode.