Immunological evaluation of urinary trypsin inhibitors in blood and urine: Role of N- & O-linked glycoproteins

Urinary trypsin inhibitors (uTi) suppress serine proteases during inflammation. After liberation from proinhibitors (P-alpha-I and I-alpha-I) by the white blood cell (WBC) response, uTi readily pass through the kidneys into urine. A key uTi, bikunin, is attached to O-linked and N-linked glycoconjugates. Recently, uTi inhibitors, called uristatins, were found to lack the O-linked glycoconjugates. Monoclonal antibodies were produced using purified uristatin and screened for binding differences to uristatin, bikunin, P-α-I, and I-α-I. Antibody-binding patterns were characterized using immunoaffinity binding onto protein-chip surfaces and analysis by Surface Enhanced Laser Desorption/Ionization mass spectrometry (SELDI), using specimens from patients and from purified uTi standards. Antibodies were developed and used in an enzyme-linked immunosorbent assay (ELISA) method for uTi measurement in urine and plasma specimens. ELISA was performed on specimens from normal, presumed healthy, controls and from patients who had been screened for inflammation using a high sensitivity C-reactive protein (CRP) test and a complete blood count (CBC). Polyclonal antibody against uTi showed cross-reactivity with the Tamm–Horsfall protein (THP) and with proinhibitors. Screening of anti-uTi monoclonal antibodies (Mab) revealed antibodies that did not cross-react with either of the above, thus providing a tool to measure both uristatin and bikunin in urine with Mab 3G5 and in plasma with Mab 5D11. The monoclonal antibody 5D11 cross-reacts with specific N-linked glycoconjugates of uristatin present in plasma. In ca 96% of healthy adults, uTi were present at <12 mg/l in urine and <4 mg/l in plasma. We also found that patients with an inflammation and a CRP of >2.0 mg/l had higher urinary concentrations of uTi than the control population in every subject. Free uristatin and bikunin pass readily into urine and are primarily bound to heavy chains that constitute the proinhibitor form in plasma.     

Base-flipping dynamics in a DNA hairpin processing reaction

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein–DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.     

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection.     

Gamma Study of pH, Nitrite, and Salt Inhibition of Aeromonas hydrophila

The gamma hypothesis states that there are no interactions between antimicrobial environmental factors. The time to growth of Aeromonas hydrophila challenged with pH, NaNO₂, and salt combinations at 30°C was investigated. Data were examined using a model based on the gamma hypothesis (the gamma model), which takes into account variance-stabilizing transformations and which gives biologically relevant parameters. At high concentrations of NaNO₂ and at pHs of >6.0, the antimicrobial action of the nitrite ion has a strong influence (MIC = 2,033 mg liter⁻¹), whereas at pHs of <6, nitrous acid is dominant (MIC = 1.5 mg liter⁻¹). This change is not due to a “synergy” between pH and the nitrite ion but is due to the shift in the equilibrium concentrations of nitrous acid and nitrite in solution caused by pH. In combination with salt, the parameters found for the action of Na nitrite were identical to those found when it was examined in isolation. Therefore, pH, NaNO₂, and salt act independently on the growth of A. hydrophila. By expanding the gamma model with a cardinal temperature model, the results of fitting the model of Palumbo et al. (J. Food Prot. 54:429-435, 1994) to randomly produced environmental conditions could be reproduced, suggesting that temperature also has an independent effect.     

Formation of Tellurium Nanocrystals during Anaerobic Growth of Bacteria That Use Te Oxyanions as Respiratory Electron Acceptors

Certain toxic elements support the metabolism of diverse prokaryotes by serving as respiratory electron acceptors for growth. Here, we demonstrate that two anaerobes previously shown to be capable of respiring oxyanions of selenium also achieve growth by reduction of either tellurate [Te(VI)] or tellurite [Te(IV)] to elemental tellurium [Te(0)]. This reduction achieves a sizeable stable-Te-isotopic fractionation (isotopic enrichment factor [] = −0.4 to −1.0 per ml per atomic mass unit) and results in the formation of unique crystalline Te(0) nanoarchitectures as end products. The Te(0) crystals occur internally within but mainly externally from the cells, and each microorganism forms a distinctly different structure. Those formed by Bacillus selenitireducens initially are nanorods (~10-nm diameter by 200-nm length), which cluster together, forming larger (~1,000-nm) rosettes composed of numerous individual shards (~100-nm width by 1,000-nm length). In contrast, Sulfurospirillum barnesii forms extremely small, irregularly shaped nanospheres (diameter < 50 nm) that coalesce into larger composite aggregates. Energy-dispersive X-ray spectroscopy and selected area electron diffraction indicate that both biominerals are composed entirely of Te and are crystalline, while Raman spectroscopy confirms that they are in the elemental state. These Te biominerals have specific spectral signatures (UV-visible light, Raman) that also provide clues to their internal structures. The use of microorganisms to generate Te nanomaterials may be an alternative for bench-scale syntheses. Additionally, they may also generate products with unique properties unattainable by conventional physical/chemical methods.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.     

Results of satellite tagging of Atlantic bluefin tuna, <Emphasis Type="Italic">Thunnus thynnus</Emphasis>, off the coast of Ireland

Pop-up satellite archival tags were attached to six Atlantic bluefin tuna (Thunnus thynnus) off the west coast of Ireland in autumn 2003 and 2004. The satellite tags measured pressure, ambient temperature and light for the term of deployment. Radio pop-up satellite endpoint positions, light and sea surface temperature estimations of geolocation indicate that two fish tagged minutes apart off the coast of County Donegal, migrated to the eastern and western Atlantic Ocean over the following 8 months. The two fish were 5218 km apart at the termination of the experiment. After tagging in September and popping up the following March and April, one fish had traveled to the western Atlantic while the other was located in the waters off the southwest coast of Portugal. A third fish tagged off the coast of County Donegal in October 2004 moved into the Mediterranean Sea and was caught by a fishing vessel southeast of Malta on 11 June 2005. The results link bluefin tuna feeding on European foraging grounds with known eastern breeding regions and western Atlantic waters.     

Metal Complexes of 3-Thiophene Carboxamides Containing a Pyridine Ring

Complexes of Zn(II), Cu(II) and Co(II) with either N-(2-methylpyridyl)-3-thienyl-alkyl-carboxamide or N-(2-pyridyl)-3-thienylalkyl-carboxamide groups have been prepared and characterized. Crystal structures of ten new complexes are reported and discussed. N-(2-Methylpyridyl)-3-thienyl-alkyl-carboxamide exhibits both uni- and bidentate behavior. With all ligands, bidentate complexation is through the carbonyl oxygen and pyridine nitrogen atoms (O, N) and the amide nitrogen atom remains protonated. The electrochemical behavior and the infrared spectra of selected complexes are discussed.     

Geographic patterns in the distribution of Palearctic songbirds

A database was created of digitized equal area distribution maps of 3,036 phylogenetic species of Palearctic songbirds. Biogeographic patterns are reported for two data sets: (1) including all passeriform bird species reported as breeding within the boundaries of our study map, (2) passeriform species restricted in their distribution to our study region, thus excluding the partly extra-limital taxa. With respect to the data set excluding partly extra-limital taxa, the average range size is 238 grid cells (grid cell area: 4,062 km²). Analysis of the geographic distribution of species richness for the full data set showed several hotspot regions, mostly located in mountainous areas. The index of range-size rarity identified similar hotspot regions as that for species richness, albeit that the range-size rarity de-emphasized the central Siberian hotspot. Range-size rarity hotspots that are not evident on the measure of species richness concern a great number of islands. Much more prominent on the index of range-size rarity are the Atlas Mountains of northern Africa, the Jabal al Akhdar region in NE Libya, and the eastern border of the Mediterranean. Restricting the analysis of geographic variation to the 25% of the species with smallest ranges resulted in a greatly simplified pattern of hotspots. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.