Photon correlation spectroscopy of human IgG

The translational diffusion coefficient D_20,w⁰, of monomeric human immunoglobulin G (IgG) has been studied by photon-correlation spectroscopy as a function of pH and protein concentration. At pH 7.6, we find D_20,w⁰=3.89×10^–7±0.02 cm²/sec, in good agreement with the value determined by classic mehods. This value corresponds to an effective hydrodynamic radius R, of 55.1±0.3 Å. As pH is increased to 8.9; with the same ionic strength, the molecule appears to expand slightly (3.5% increase in hydrodynamic radius). The concentration dependence of the IgG diffusion constant is interpreted in terms of solution electrostatic effects and shows that long-range repulsive interactions are negligible in the buffer used. The diffusion coefficient for dimeric IgG has also been determined to be D_20,w=2.81×10^–7±0.04 cm²/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 Å. For light-scattering studies of protein molecules in the dimension range of 5–10 nm (M_r=10⁵–10⁷) we find monomeric horse spleen ferritin well suited as a reference standard. Ferritin is a spherical molecule with a hydrodynamic radius R of 6.9±0.1 nm and is stable for years in our standard Tris-HCl-NaCl buffer even at room temperature.     

Long-Term Drug Treatment of Obesity in a Private Practice Setting

ATKINSON, RICHARD L, ROY C BLANK, DONALD SCHUMACHER, NIKHIL V DHURANDHAR, DOUGLAS L RITCH. Long-term drug treatment of obesity in a private practice setting. This study evaluated the long-term efficacy and safety of the combination of phentermine and fenfluramine for the treatment of obesity in a private practice setting. A total of 1388 consecutive, qualified patients presenting to a private general internal medicine practice in Charlotte, NC, were enrolled with eligibility criteria including: age 18 years to 60 years, 20% over “desirable” bodyweight or body mass index <27, no serious medical or psychiatric disease, and no contraindications to drug therapy. Patients were instructed in diet, exercise, and behavior modification techniques and received phentermine (15 mg/day to 30 mg/day) and fenfluramine (20 mg/day to 60 mg/day) continuously for over 3 years. Average duration of treatment was 15. 9 months, and average weight loss at the last visit was 11. 6 kg, or 11. 7% of initial bodyweight. For patients completing 1 year of drug treatment, mean weight loss was 16. 5 kg, or 16% of initial weight. Weight loss persisted for 2 years, but partial regain was seen at 3 years. The dropout rates were 18% at 6 months, 39% at 1 year, 68% at 2 years, and 78% at 3 years. At 1 year, blood pressure of hypertensive patients fell from 151/95 mm Hg to 127/78 mm Hg, and serum cholesterol and triglycerides of hyperlipidemic patients fell by 0. 750 mmol/L (29 mg/dL) and 0. 937 mmol/L (83 mg/dL), respectively. Adverse events were modest. We conclude that, in a private practice setting, long-term treatment of obesity with the combination of phentermine, fenfluramine, and a weight maintenance program is generally safe and effective. More research is needed to determine efficacy and safety for longer than 3 years.     

Gold from the sea: marine compounds as inhibitors of the hallmarks of cancer

Cancer is one of the most deadly diseases in the world. Although advances in the field of chemo-preventive and therapeutic medicine have been made regularly over the last ten years, the search for novel anticancer treatments continues. In this field, the marine environment, with its rich variety of organisms, is a largely untapped source of novel compounds with potent antitumor activity. Although many reviews of marine anticancer compounds have been published, we focus here on selected marine compounds that act on the six hallmarks of cancer presented namely self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replication, sustained angiogenesis and tissue invasion and metastasis.     

Galectin-3 regulates integrin alpha2beta1-mediated adhesion to collagen-I and -IV

Galectins are a taxonomically widespread family of galactose-binding proteins of which galectin-3 is known to modulate cell adhesion. Using single cell force spectroscopy, the contribution of galectin-3 to the adhesion of Madin-Darby canine kidney (MDCK) cells to different extracellular matrix proteins was investigated. When adhering to collagen-I or -IV, some cells rapidly entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Galectin-3-depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin alpha(2)beta(1), as it was not observed when cells adhered to extracellular matrix substrates by other integrins. The adhesion phenotype of galectin-3-depleted cells was mimicked in a galactoside-deficient MDCK cell line and could be complemented by the addition of recombinant galectin-3. We propose that galectin-3 influences integrin alpha(2)beta(1)-mediated adhesion complex formation by altering receptor clustering.     

Morphological diversity of Ethiopian barley (Hordeum vulgare L.) in relation to geographic regions and altitudes

A total of 199 germplasm accessions collected from 10 administrative regions of Ethiopia and four released cultivars, which were used for estimating of error variance, of barley in Ethiopia were field evaluated for nine agronomic traits at Holetta and Bekoji Agricultural Research Centers of Ethiopia during the 2006 main cropping season using non-replicated augmented design plots consisting of four incomplete blocks. The objectives were to assess the extent and pattern of morphological variation in the barley accessions with respect to regions and altitude of collection, to classify the genotypes tested into relatively homogenous groups and to identify the major traits contributing to the overall observed diversity in the germplasm. Genotype variance estimate of regions and altitudes indicated wide variation among accessions depending on the traits involved. The presence of high morphological variation within regions and altitudes particularly above 2000 m a.s.l. indicated the potential of each region and high altitude zones for barley improvement and conservation in the country. The clustering of accessions did not show grouping on the basis of regions of origin. Traits like thousand kernel weight, plant height, days to head and days to maturity accounted for most of the gross variance among the barley accessions and played role in differentiating accessions collected from different regions and altitude classes into principal components. In general because of environmental factors on the observed morphological variation future germplasm collection should consider to explore wide geographical and climatic differences within the country.     

Decimal Place Slope,A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of Microbial Species

The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of ¹³C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [¹³C₆]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of ¹³C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful component of qualitative and quantitative proteome studies (1). Incorporation of heavy isotopes can be used to analyze microbial processes such as turnover rates and also to help to establish structure-function relationships within microbial communities. Stable isotope probing (SIP¹) techniques based on DNA-SIP (2) and RNA-SIP (3) have been used for this purpose previously. With the introduction of protein-SIP (4), the need for an accurate alternative method for calculating label incorporation into biomolecules arose. Protein-SIP has several advantages compared with DNA/RNA-SIP, the most important being its capacity to detect dynamic levels of incorporation, whereas only labeled or unlabeled states can be categorized by means of DNA/RNA-SIP because of the need to separate ¹³C-DNA/RNA by density gradient centrifugation. Quantitative analysis of ¹³C incorporation levels is of the utmost importance, especially when unraveling carbon fluxes through either microbial communities or food webs with different trophic levels.In contrast to the incorporation of isotopically labeled amino acids, which is often used in quantitative proteomics (5), metabolic labeling by growth substrates and nutrients (e.g. salts) is often imperfect and makes the processing of mass spectrometry (MS) data difficult. For example, when the incorporation of ¹³C exceeds ∼2 atom %, common database search algorithms fail to identify peptides and proteins. The problem can only be managed successfully if a stable, known degree of ¹³C incorporation can be achieved during the experiment (6). Using a low labeling efficiency of roughly 5 atom %, Huttlin et al. (6) chose the altered envelope chain for calculating the incorporation and simultaneously used the signal intensity for a quantitative comparison with the sample that had a natural abundance of ¹³C. Database approaches for peptide identification can cope only with the natural abundance of carbon isotopes; they fail if the incorporation of ¹³C significantly exceeds the natural isotope abundance or if incorporation patterns occur in unpredictable ways (7).The simplest method for determining the incorporation level is to compare the unlabeled average mass of the monoisotopic peptide with the mass of the labeled protein, as estimated by matrix-assisted laser desorption/ionization or electrospray ionization MS (8, 9). A more advanced approach for determining the isotopic mass distribution of peptides is based on the isotopic distribution of the peaks of a peptide envelope (10, 11). Here, for a given isotopomer, the incorporation efficiency is defined as the percentage of incorporated ¹³C atoms with relation to the total number of carbon atoms with the natural isotope abundance (approximately 1.01 atom % ¹³C). As a reference, the theoretical isotopic distribution of a peptide is calculated based upon an algorithm described elsewhere (12). The isotope distribution of both unlabeled and labeled peptides can subsequently be used to calculate the incorporation level. For this method, an Excel spreadsheet (ProSIPQuant.xls) was developed (4). A similar approach, also based on the calculation of isotopic distributions, has been used in other studies (7). In these studies, however, the identification of the peptides is limited to those that have unlabeled counterparts; in addition, an exact calculation can be hampered by overlapping signals coming from additional peaks with similar masses.In the present study, we describe a new way of determining the isotope incorporation level. Our method makes use of characteristic patterns in the digits after the decimal point of the peptide masses generated by high-accuracy instruments such as the linear ion trap LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). For tryptic peptides, typical regularities in the decimal places of the monoisotopic masses have been observed (13, 14). These observations have been explored in detail for theoretical and experimental data of proteins originating from Helicobacter pylori (15). As a result, a rule called the “half decimal place rule” (HDPR) was defined; it states that the decimal place is nearly half of the first digit for tryptic peptides with masses in the range of 500–1,000 Da. In other words, the exact mass of a peptide is equal to its nominal mass times ∼1.005. Because the difference between ¹²C and ¹³C is slightly greater than 1 Da, exactly 1.0033548378, the decimal places of a tryptic peptide''s mass are shifted in a regular manner by the incorporation level and lead to a significantly increased slope for the digits in the third and fourth place after the decimal point. This shift can be used to estimate the incorporation level of heavy isotopes into the protein. Detecting such shifts requires the highly accurate measurement possible with modern mass spectrometers such as the LTQ-Orbitrap, the Fourier transform ion cyclotron resonance, or the quadrupole time of flight. In this communication, we demonstrate the applicability of this approach using Pseudomonas putida ML2 proteins labeled uniformly via the consumption of [¹³C₆]benzene with five different substrate concentrations (0, 10, 25, 50, and 100 atom % of ¹³C). The ¹³C incorporation was calculated based on several labeled peptides derived from different proteins in one SDS-PAGE band. By these means, we have established a method that allows the determination of ¹³C incorporation into proteins and can be used to assess the metabolic activity of a given species within a mixed community.     

Geranic Acid Formation, an Initial Reaction of Anaerobic Monoterpene Metabolism in Denitrifying Alcaligenes defragrans

Monoterpenes with an unsaturated hydrocarbon structure are mineralized anaerobically by the denitrifying β-proteobacterium Alcaligenes defragrans. Organic acids occurring in cells of A. defragrans and culture medium were characterized to identify potential products of the monoterpene activation reaction. Geranic acid (E,E-3,7-dimethyl-2,6-octadienoic acid) accumulated to 0.5 mM in cells grown on α-phellandrene under nitrate limitation. Cell suspensions of A. defragrans 65Phen synthesized geranic acid in the presence of β-myrcene, α-phellandrene, limonene, or α-pinene. Myrcene yielded the highest transformation rates. The alicyclic acid was consumed by cell suspensions during carbon limitation. Heat-labile substances present in cytosolic extracts catalyzed the formation of geranic acid from myrcene. These results indicated that a novel monoterpene degradation pathway must be present in A. defragrans.     

Occurrence of Antimicrobial Resistance in Fish-Pathogenic and Environmental Bacteria Associated with Four Danish Rainbow Trout Farms

Surveillance of bacterial susceptibility to five antimicrobial agents was performed during a 1-year period in and around four freshwater fish farms situated along a stream in western Denmark. Besides assessing the levels of antibiotic resistance among the culturable fraction of microorganisms in fish, water, and sediment samples, two major fish pathogens (88 Flavobacterium psychrophilum isolates and 134 Yersinia ruckeri isolates) and 313 motile Aeromonas isolates, representing a group of ubiquitous aquatic bacteria, were isolated from the same samples. MICs were obtained applying a standardized agar dilution method. A markedly decreased susceptibility of F. psychrophilum isolates to most antimicrobial agents presently available for use in Danish aquaculture was detected, while the collected Y. ruckeri isolates remained largely sensitive to all therapeutic substances. Comparing the inlet and outlet samples, the increase of the antibiotic-resistant proportions observed among the culturable microflora was more pronounced and statistically significant among the motile aeromonads. High levels of individual and multiple antimicrobial resistances were demonstrated within the collected flavobacteria and aeromonads, thus indicating a substantial impact of fish farming on several groups of bacteria associated with aquacultural environments.