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In this study we examined the difference in patient INR values as measured by the POCT CoaguChek XS device and central laboratory Stago Evolution and Siemens BCS XP analyzers.This study composed of 100 warfarin therapy patients and 20 coagulation normal subjects, showed that the difference between the POCT and clinical laboratory values increased with increasing INR and was exacerbated by the use of different thromboplastin reagents by the POCT and central lab.The CoaguChek XS and on-site Stago analyzers which used human recombinant (ISI=1.01) and rabbit brain thromboplastin (ISI=1.25), respectively, showed reasonable agreement for INR3.0 (k=0.62) but significant difference for INR≥3.0 (k=0.10). In contrast, the CoaguChek XS and Siemens BCS XP, which both employed human recombinant thromboplastin (BCS ISI=1.02), showed greater agreement for the complete range INR values (INR3.0 k=0.84; INR≥3.0 k=0.70). ECAA Poller calibrant data showed the automated instruments were performing as expected, indicating that ISI calibrations were correct but insufficient to standardize the INR values for the different thromboplastin methods across the full range of measured INRs. Central lab verification of POCT INR5.0 with the Stago Evolution prevented adverse treatment events for the warfarin therapy patients in the six months preceding and following this investigation.

Human rhinovirus (HRV) is one of the most common human respiratory pathogens and is responsible for the majority of upper respiratory illnesses. Recently, a phylogeny was constructed from all known American Type Culture Collection (ATCC) HRV sequences. From this study, three HRV classifications (HRVA, HRVB, and HRVC) were determined and techniques for classifying new isolates of HRV were reported. The genetic change of this virus in specific populations over time is of great interest to understand the evolution and epidemiology of viruses. To facilitate the collections of HRV sequences over a number of years, a virology experiment was designed in which students test nasal lavage samples to look for HRV infection. Students will learn a variety of techniques including RNA isolation, cDNA synthesis, qPCR, and agarose gel electrophoresis as well as bioinformatic skills though examination of sequences from the HRV‐field isolates. Furthermore, students can look at symptom data from subjects to investigate correlations between symptom severity and factors such as stress and sleep patterns. Such information can be used to examine hypotheses regarding HRV mutation, symptom severity and epidemiology. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 6, pp. 457–458, 2011

The health benefits of ω-3 fatty acids, particularly fish oils, are well known. It is generally recommended to obtain ω-3 fatty acids from fatty fish; however, the possible contaminants in fish products may deter this. Thus, supplements of ω-3 fatty acids from plant-originated lipids may be of interest. Among these, α-linolenic acid (ALA) and stearidonic acid (SDA) have drawn attention. Thus, the purpose of this research was to test the possible conversion of these fatty acids, particularly SDA, to eicosapentaenoic acid (EPA) using a human hepatoma cell line, HepG2 cells. In addition, even though ω-3 fatty acids have been shown to reduce hyperlipidemia, the effects on lipoprotein metabolism are relatively inconsistent. Thus, we also tested ω-3 fatty acids along with SDA on apolipoprotein A and B (apo A and B) secretion from this cell line. The results obtained suggest that both ALA and SDA were converted to EPA and they increased apo B secretion, while having no effect on apo A secretion, similar to the effects of EPA and docosahexaenoic acid. Thus, it may be concluded that SDA can be a possible candidate for use as a precursor for EPA and its effect on lipoproteins was similar to that of EPA. PRACTICAL APPLICATIONS This study suggests the possible use of stearidonic and α-linolenic acids from plant-originated fat as precursors for eicosapentaenoic acid, promoting health benefits as alternatives for fish consumption.

In previously studies, we showed that tumor necrosis factor-α (TNF-a), a cytokine involved in a variety of biologically important events, is partially involved in the effects of conjugated linoleic acid (CLA). In this report, we further tested the effects of individual CLA isomers on serum TNF-a concentration along with other biological markers in mice. The animals were fed experimental diets (control, 0.5% CLA-mixed isomer, 0.25% cis-9, trans-11 CLA or 0.25% trans-10, cis-12 CLA) for 5 days and were then challenged with endotoxin. Both cis-9, trans-11 and trans-10, cis-12 CLA isomers reduced serum TNF-a levels compared to control. CLA had no effect on the other biological markers examined. These results suggest possible early involvement of CLA in immune and/or inflammatory responses, followed by reduction of body fat. Its effect on TNF-a helps explain in part how CLA modulates other biological functions such as immune response, insulin responses, atherosclerosis and cancer.

The elongated form of conjugated linoleic acid (CLA), conjugated eicosadienoic acid (CEA, conj. 20:2delta(c11,t13/t12,c14)), was generated from CLA by liver microsomal fractions. Subsequent testing showed that dietary CEA significantly reduced body fat, and increased lean mass similar to CLA when compared to controls. CEA also decreased lipoprotein lipase activity and triacylglyceride, and increased glycerol release in 3T3-L1 adipocytes, correlated with the trans-12,cis-14 isomer, but CEA required a longer incubation period than cells treated with CLA. Based on the fact that CEA fed animals had CLA in tissue, we suggest that the effect of CEA is due to the CLA converted from CEA in the system. The delta-6 desaturated and elongated form of trans-10,cis-12 CLA (conjugated eicosatrienoic acid, CETA, conj. 20:3delta(c8,t12,c14)) inhibited LPL activity and increased glycerol release but was less active than trans-10,cis-12 CLA or CEA. The 21-carbon conjugated fatty acid, conjugated heneicosadienoic acid (CHDA, conj. 21:2delta(c12,t14/c13,t15)), was not active on LPL inhibition, triacylglyceride, or glycerol release in 3T3-L1 adipocytes. We also provide evidence that CLA was metabolized to conjugated dodecadienoic acid (conj. 12:2delta(c3,t5/t4,c6)). In addition, there were indications of the presence of conjugated tetradecadienoic acid (conj. 14:2delta(c5,t7/t6,c8)), suggesting that CLA can be metabolized through fatty acid beta-oxidation. This is the first work to report the presence of conjugated 12 and 14 carbon fatty acids, originated from CLA, and the biological activities of CEA, CETA and CHDA.

Conjugated linoleic acid (CLA) reduces body fat in part by inhibiting the activity of heparin-releasable lipoprotein lipase (HR-LPL) activity in adipocytes, an effect that is induced by the trans-10,cis-12 CLA isomer. In this study we used a series of compounds that are structurally related to CLA (i.e., CLA cognates) to investigate the structural basis for this phenomenon. None of the 18:1 CLA cognates that were tested, nor trans-9,cis-12 18:2, cis-12-octadecen-10-ynoic acid (10y,cis-12) or 11-(2'-(n-pentyl)phenyl)-10-undecylenic acid (designated P-t10), exhibited any significant effect on HR-LPL activity. Among the CLA derivatives (alcohol, amide, and chloride) that were tested, only the alcohol form inhibited HR-LPL activity, although to a lesser extent than CLA itself. In addition, intracellular TG was reduced only by trans-10,cis-12 CLA and the alcohol form of CLA. Hence it appears that the trans-10,cis-12 conjugated double bond in conjunction with a carboxyl group at C-1 is required for inhibition of HR-LPL activity, and that an alcohol group can partially substitute for the carboxyl group. We also studied glycerol release from the cells, observing that this was enhanced by trans-10 18:1, trans-13 18:1, cis-12 18:1, cis-13 18:1, P-t10 but was reduced by cis-9 18:1, the alcohol and amide forms of CLA or 10y,cis-12. Accordingly the structural feature or features involved in regulating lipolysis appear to be more complex. Despite enhancing lipolysis in cultured 3T3-L1 adipocytes, trans-10 18:1 did not reduce body fat gain when fed to mice.

Two experiments were conducted. In Experiment 1, 8-wk-old mice were fed control diet or diet supplemented with 0.5% conjugated linoleic acid (CLA) to study the effect of CLA on body composition (CLA: 40.8-41.1% c-9,t-11 isomer, 43.5-44.9% t-10,c-12 isomer). The data for CLA-fed mice vs. controls described parallel but significantly distinct responses for both absolute and relative changes in body fat mass (reduced in CLA-fed mice) and for relative changes in whole body protein and whole body water (both of which were increased in CLA-fed mice). In the CLA-fed mice, the effect on whole body protein appeared to precede the reduction in body fat mass. In Experiment 2, weanling mice were fed control diet or diet supplemented with 0.5% CLA for 4 wk (test group), at which time all mice were fed control diet devoid of added CLA. The test group exhibited significantly reduced body fat and significantly enhanced whole body water relative to controls at the time of diet change. Time trends for changes in relative body composition were described by parallel lines where the test group exhibited significantly less body fat but significantly more whole body protein, whole body water, and whole body ash than controls. Tissue CLA levels declined following the withdrawal of CLA from the diet. In skeletal muscle of mice fed CLA-supplemented diet, the t-10,c-12 isomer was cleared significantly faster than the c-9,t-11 CLA isomer.

The effects of conjugated linoleic acid (CLA) on body composition were investigated. ICR mice were fed a control diet containing 5.5% corn oil or a CLA-supplemented diet (5.0% corn oil plus 0.5% CLA). Mice fed CLA-supplemented diet exhibited 57% and 60% lower body fat and 5% and 14% increased lean body mass relative to controls (P < 0.05). Total carnitine palmitoyltransferase activity was increased by dietary CLA supplementation in both fat pad and skeletal muscle; the differences were significant for fat pad of fed mice and skeletal muscle of fasted mice. In cultured 3T3-L1 adipocytes CLA treatment (1 x 10(-4)M) significantly reduced heparin-releasable lipoprotein lipase activity (-66%) and the intracellular concentrations of triacylglyceride (-8%) and glycerol (-15%), but significantly increased free glycerol in the culture medium (+22%) compared to control (P < 0.05). The effects of CLA on body composition appear to be due in part to reduced fat deposition and increased lipolysis in adipocytes, possibly coupled with enhanced fatty acid oxidation in both muscle cells and adipocytes.

Previously it was reported that 4-hydroxy-2 (or 5)-ethyl-5 (or 2)-methyl-3(2H)-furanone (HEMF), a characteristic flavour component of Japanese-style fermented soy sauce that exhibits antioxidant activity, inhibits benzo[a]pyrene-induced forestomach neoplasia in mice. The antioxidant and anticarcinogenic activities of other structurally similar soy sauce flavour components are now reported. 4-Hydroxy-5-methyl-3(2H)-furanone (HMF) and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) were found to be antioxidants. In particular, HMF and HDMF (as well as HEMF) reduced hydrogen peroxide concentration in human polymorphonuclear leucocytes stimulated by arachidonic acid or 12-O-tetradecanoylphorbol-13-acetate. HMF and HDMF were administered individually in semipurified diet to female ICR mice previously treated with benzo[a]pyrene (1.5 mg/wk, orally for 4 wk) to initiate forestomach neoplasia. The mice were killed at 30 wk of age. Both furanones reduced forestomach neoplasms, with HDMF exhibiting more potency. The data indicate that HDMF and HMF, like HEMF, inhibit carcinogenesis in this system by acting at the post-initiation stage.

The pharmacokinetics of 5-fluorouracil (5FU) were studied in vivo in patients with discrete tumors and in rabbits bearing VX2 tumors by using 19F NMR spectroscopy. The human studies were conducted in a 1.5-T Magnetom magnetic resonance imager (Siemens), and the rabbit studies were conducted in a 4.7-T GE/Nicolet 33-cm bore magnet. Free 5FU was detected in the tumors of four of the six patients and in all VX2 tumors but not in normal rabbit tissues. No other metabolites were seen in these tumors, contrary to the extensive catabolism we had previously documented using 19F NMR spectroscopy in both human and animal livers. The tumor pool of free 5FU in those human tumors that trapped 5FU was determined to have a half-life of 0.4-2.1 hr, much longer than expected and significantly longer than the half-life of 5FU in blood (5-15 min), whereas the half-life of trapped 5FU in the VX2 tumors ranged from 1.05 to 1.22 hr. In this initial experience, patient response to chemotherapy may correlate with extent of trapping free 5FU in the human tumors. These studies document that NMR spectroscopy is clinically feasible in vivo, allows noninvasive pharmacokinetic analyses at a drug-target tissue in real time, and may produce therapeutically important information at the time of drug administration. Demonstration of the trapping of 5FU in tumors provides both a model for studying metabolic modulation in experimental tumors (in animals) and a method for testing modulation strategies clinically (in patients).

The trans-10,cis-12 isomer of conjugated linoleic acid (CLA) has been shown to reduce body fat gain in mice. However, the underlying molecular mechanism is not well characterized. Here we report evidence that trans-10,cis-12 (t10c12) CLA inhibits preadipocyte differentiation. Treating differentiating 3T3-L1 preadipocytes with t10c12 CLA and conjugated nonadecadienoic acid (CNA, a 19-carbon CLA cognate) resulted in decreased intracellular triglyceride accumulation and mRNA levels of the adipogenic gene fatty acid synthase and adipocyte lipid binding protein. T10c12 CLA and CNA also reduced protein levels of adipocyte transcription factors, peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Similarly, CLA reduced body fat gain and significantly inhibited the expression of PPAR gamma and its downstream target lipoprotein lipase in mouse adipose tissue. These observations indicate that CLA decreases body fat gain in part by inhibiting the differentiation of preadipocytes.

We investigated the effects of conjugated linoleic acid (CLA) preparations, which were enriched for the cis-9,trans-11 CLA isomer or the trans-10,cis-12 CLA isomer, on body composition in mice. Body composition changes (reduced body fat, enhanced body water, enhanced body protein, and enhanced body ash) were associated with feeding the trans-10,cis-12 CLA isomer. In cultured 3T3-L1 adipocytes, the trans-10,cis-12 isomer reduced lipoprotein lipase activity, intracellular triacylglycerol and glycerol, and enhanced glycerol release into the medium. By contrast, the cis-9,trans-11 and trans-9,trans-11 CLA isomers did not affect these biochemical activities. We conclude that CLA-associated body composition change results from feeding the trans-10,cis-12 isomer.

The amidase activity of human alpha-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 degrees C. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 degrees C. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KCl, and MgCl2 show that human alpha-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human alpha-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human alpha-thrombin and its structurally perturbed derivatives.

Conjugated dienoic derivatives of linoleic acid (referred to by the acronym CLA) constitute a newly recognized class of anticarcinogenic fatty acids. Of the eight major CIA isomere, the cis-9, trans-11 isomer alone is incorporated into phospholipid and may to be the most biologically relevant isomer. CLA exhibits potent antioxidant activity; evidence is presented indicating that CLA acts both as an in vitro and in vivo antioxidant. The formation of CLA in foods, and its possible biological significance in cell membranes, is discussed.

The crystal structure of η 5 -C 5 H 5 (CO) 3 MoHgCl has been determined from single crystal X-ray data collected by counter methods. The compound crystallizes in space group P42 1 c with 8 molecules per unit cell and lattice constants of a  12.014(4), c  15.322(7). Full-matrix least squares refinement gave discrepancy factors R 1  0.058 and R 2  0.062 for 854 data with 1 3σ (1). The critical bond lengths and angles are MoHg  2.673(3)A, HgCl  2.437(8)A, and MoHgCl  160.0(2)°. Crystals of η 5 C 5 H 5 (CO) 3 WHgBr were shown to be isomorphous with lattice constants of a  12.068(4)A and c  15.643(7)A.

A 13 C NMR investigation of terminally substituted normal butenes, pentenes, and hexenes is reported. These studies include data on one, two, and three bond 13 C— 199 Hg coupling constants as well as on changes in the chemical shifts of all carbons as a function of the electronegativity of the substituent. Attempts to correlate the chemical shifts of the olefinic carbons with metal—double bond interactions were inconclusive; however, large effects were observed on the chemical shifts of the olefinic carbon atoms when dipentenylzinc was complexed with 2,2′-bipyridyl. These shifts are attributed to the large anisotropic magnetic field associated with the aromatic rings and the proximity of the olefinic carbon atoms to these rings.

Hydrostatic pressure has been shown to inhibit protein synthesis in Escherichia coli by inhibiting amino acid activation and polypeptide synthesis in cell-free systems. Pressure may decrease translational ambiguity by suppressing any non-specific activity of phenylalanyl-tRNA synthetase and by preferentially decreasing the stability of leucyl-tRNA.

A 199Hg Fourier Tranformation NMR study has been carried out on Hg[M(CO)3C5H5]2 (M = Cr, Mo, W), [C5H5(CO)3M]HgCl, and [C5H5(CO)3W]HgX (X = Cl, Br, I, SCN). The 199Hg chemical shifts are −80, +115 and −348 ppm respectively for the symmetrical compounds, −542, −617, and −997 ppm for the chloride and −1200, −1529 and −924 ppm for the Br, I, SCN-tungsten derivatives respectively. The 199Hg chemical shifts for several other mercury derivatives are reported for comparison. All chemical shifts are relative to 90% HgMe2/10% internal lock C6F6 with positive values indicating decreasing shielding. The J199Hg-183W values are 151, 706, 690, 630 and 684 Hz for the symmetrical compound and the Cl, Br, I and SCN derivatives respectively. The factors which may influence these parameters are discussed briefley.

The crystal and molecular structure determinations of 2,2,4,4,6,6,8,8-octamethyl-2,4,6,8-tetrasila-1,5-mercuracyclooctane. Hg 2 Si 4 C 10 H 28 (I) and of bis-(triphenylsilyl)mercury, (Ph 3 Si) 2 Hg (II), are reported. The structures have been determined from single-crystal X-ray data collected by counter methods. Both molecules crystalize in the space group P 1 with one centrosymmetric molecule per unit cell. Each structure contains linear SiHgSi groups, with mercury-silicon distances of 2.490(4) A in I and 2.503(4) A II. In compound I the SiHgSi groups are linked by methylene bridges which form an eight member ring in the chair conformation. The cell dimensions for compound I are a 6.277(2), b 8.408(2), c 9.274(4) A, α 92.75(3), β 94.79(3) and γ 100.14(2)° with R 1 0.062 for 1809 observed reflections. The cell dimensions for compound II are a 9.999(4), b 11.727(8), c 7.654(5) A, α 99.87(5), β 115.35(4) and γ 98.41(4)°, with R 1 0.081 for 2394 observed reflections.

The temperature and hydrostatic pressure maxima for growth of the marine psychrophilic bacterium, Vibrio marinus MP-4, were 20C and 425 atm. The effects of pressures of 200, 400, 500, and 1,000 atm at 15C on synthesis of net protein, RNA, and DNA by growing cells of V. marinus MP-4 were determined. A pressure of 1,000 atm completely inhibited protein and DNA synthesis but allowed RNA synthesis decreased for approximately 60 min and then resumed the 1-atm rates, whereas DNA synthesis was unaffected. Pressures of 400 and 500 atm immediately lowered the rate of protein synthesis, whereas RNA synthesis was unaffected by 400 atm. At 500 atm, RNA synthesis continued at the 1-atm rate for 30 min and then shifted to a lower rate. Synthesis of DNA, at 400 and 500 atm, continued at the 1-atm rate for approximately 60 min after pressurization, then gradually shifted to lower rates. The data suggest that the primary effect of pressures from 400 to 500 atm was to lower the rate of protein synthesis, which in turn may have lowered the rates of RNA and DNA synthesis.

Vibrio marinus, an obligately psychrophilic marine bacterium, deaminated nine of 17 amino acids tested with both L-glutamine and L-serine displaying the greatest deamination rates.

Hydrostatic pressure inhibits the binding of aminoacyl transfer RNA to ribosomes in a cell-free system from Escherichia coli , and has an even greater inhibitory effect on peptide bond formation. Evidence that pressure decreases the stability of the messenger RNA-ribosome complex is also presented.

Filtrates from microbial heterotrophic activity assay experiments in which tritiated glucose was used as a radioactively labeled substrate were collected, and their radioactivities were determined. These filtrates were subsequently evaporated to dryness to remove tritiated water generated by metabolism of the labeled glucose; the residue was suspended to original volume with distilled water, and the tritium levels were again assayed. In the water samples assayed, the amount of tritium label retained by the microorganism was about 75% of that respired.

The contribution of attached and free-floating bacteria to the bacterial numbers and heterotrophic uptake in 44 diverse aquatic environments was studied. A factor analysis reduced the variability of the raw data base to three major factors explaining 53.6% of total variance. These factors were (i) salinity, (ii) heterotrophic uptake, and (iii) particulate load. A cluster analysis categorized the 44 habitats into five distinct environmental types based on these three factors. There was no significant pattern in the distribution of attached versus free-floating bacteria when assessed by epifluorescent microscopy. However the contribution of attached bacteria to the uptake of an amino acids mix was reduced in marine waters. Heavy particulate loads resulted in an increased percentage uptake of amino acids and glucose from the attached bacteria. Uptake response was found to be substrate specific especially in oliogotrophic freshwater. Amino acid uptake was more associated with the attached fraction, whereas glucose uptake was mediated more by the free-floating fraction.

Increasing amounts of high-specific-activity tritiated organic compounds were added to samples of several natural waters such that in situ substrate concentrations might be approximated. The uptake responses by the native heterotrophic microflora suggested that (i) heterotrophic populations metabolize the added nutrients, but (ii) these responses are not necessarily a reflection of Michaelis-Menten enzyme kinetics. The uptake kinetics appeared to be due to dilution of the naturally occurring metabolite by added radioactive substrate and physiological responses of the microflora to organic enrichment.

The effects on growth and net protein, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) synthesis of alternately pressurizing and depressurizing exponentially growing cells of Escherichia coli B/r and Vibrio marinus MP-1 between 1 atm and 544 atm of hydrostatic pressure were studied. The application of 544 atm to these 1 atm exponentially growing cultures caused an almost immediate cessation or lowering in the rates of cell division, absorbance changes, and net protein and RNA synthesis. The cells of E. coli B/r, however, in some manner adapted to 544 atm, since cell division, absorbance, and synthesis of protein and RNA, within minutes, resumed exponential increases but at much lower rates. V. marinus MP-1 did not display this pressure adaption upon the first application of 544 atm. Upon pressure release both cultures resumed the 1-atm exponential increases with respect to cell division and absorbance, and net protein and RNA synthesis. A second application of 544 atm, however, had a less drastic influence on cell division and the rates of increase of absorbance and net protein and RNA synthesis. Both cultures immediately shifted to lower synthetic rates. There was no immediate effect upon DNA synthesis in these two bacteria upon application of 544 atm, but this synthetic process slowed and ceased with time. The data indicate a reversible sensitivity to 544 atm in the order protein > RNA > DNA synthesis and that the cells can in some fashion adapt to pressure resistance.