Influence of Zn<Superscript>2+</Superscript>, Co<Superscript>2+</Superscript> and dimethylbenzimidazole on vitamin B<Subscript>12</Subscript> biosynthesis by <Emphasis Type="Italic">Pseudomonas denitrificans</Emphasis>

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B₁₂ biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn²⁺, Co²⁺ and DMBI on vitamin B₁₂ production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B₁₂. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B₁₂ production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.     

Vitamin B<Subscript>12</Subscript> as a carrier for targeted platinum delivery: in vitro cytotoxicity and mechanistic studies

It is attractive to use vitamin B₁₂ as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B₁₂ by fast proliferating cells. The basic {B₁₂–CN–Pt^II} conjugates are recognized by intracellular enzymes and converted to coenzyme B₁₂ in an enzymatic adenosylation assay. The reductive adenosylation of {B₁₂–CN–Pt^II} conjugates leads to the release of the Pt^II complexes; thus, {B₁₂–CN–Pt^II} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin–B₁₂ conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B₁₂–CN–Pt^II} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding Pt^II species. Kurnakov tests and coordination of 2′-deoxyguanosine or GMP to the released Pt^II complexes allowed isolation and characterization of Pt^II complexes as released during enzymatic adenosylation. The biological activity of these Pt^II complexes was evaluated. Since the cleaved Pt^II complexes show cytotoxicity, the {B₁₂–CN–Pt^II} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC₅₀ between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B₁₂–CN–Pt^II} concentration, comparison with pure cisplatin is of limited use. We could show that the Pt^II complexes cleaved from B₁₂ exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B₁₂ free media showed a dependence on the addition of transcobalamin II for B₁₂–Pt(II) conjugates.     

Inflammation causes tissue-specific depletion of vitamin B<Subscript>6</Subscript>

Previously we observed strong and consistent associations between vitamin B₆ status and several indicators of inflammation in patients with rheumatoid arthritis. Clinical indicators, including the disability score, the length of morning stiffness, and the degree of pain, and biochemical markers, including the erythrocyte sedimentation rate and C-reactive protein levels, were found to be inversely correlated with circulating vitamin B₆ levels. Such strong associations imply that impaired vitamin B₆ status in these patients results from inflammation. In the present study we examined whether inflammation directly alters vitamin B₆ tissue contents and its excretion in vivo. A cross-sectional case-controlled human clinical trial was performed in parallel with experiments in an animal model of inflammation. Plasma and erythrocyte and pyridoxal 5'-phosphate concentrations, urinary 4-pyridoxic acid excretion, and the activity coefficient of erythrocyte aspartate aminotransferase were compared between patients and healthy subjects. Adjuvant arthritis was induced in rats for investigating hepatic and muscle contents as well as the urinary excretion of vitamin B₆ during acute and chronic inflammation. Patients with rheumatoid arthritis had low plasma pyridoxal 5'-phosphate compared with healthy control subjects, but normal erythrocyte pyridoxal 5'-phosphate and urinary 4-pyridoxic acid excretion. Adjuvant arthritis in rats did not affect 4-pyridoxic acid excretion or muscle storage of pyridoxal 5'-phosphate, but it resulted in significantly lower pyridoxal 5'-phosphate levels in circulation and in liver during inflammation. Inflammation induced a tissue-specific depletion of vitamin B₆. The low plasma pyridoxal 5'-phosphate levels seen in inflammation are unlikely to be due to insufficient intake or excessive vitamin B₆ excretion. Possible causes of decreased levels of vitamin B₆ are discussed.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

Effect of chemical and microbial vitamin B<Subscript>12</Subscript> analogues on production of vitamin B<Subscript>12</Subscript>

Strain improvement by genetic manipulation or optimization of fermentation conditions for overproduction of vitamin B₁₂ has a drawback due to feed back inhibition. To resist the feed back inhibition by analogues of vitamin B₁₂ in Propionibacterium freudenrechii subsps. shermanii (OLP-5), we have tested with microbially separated B₁₂ analogues from three different strains. Microbial analogues were differentiated from commercially available vitamin B₁₂ by high pressure liquid chromatography and spectrophotometric method. An analogue isolated from NRRL-B-4327 was shown to increase vitamin B₁₂ concentration from 18.53 ± 0.15 to 31.67 ± 0.58 mg/l in OLP-5 strain. The presence of chemical analogue (ICH₂ Co(DH)₂ (H₂Py)₄) increased vitamin B₁₂ production from 16.13 ± 0.15 to 18.53 ± 0.15 mg/l in OLP-5. These findings revealed that addition of B₁₂ analogues in fermentation media have developed strain resistance to feed back inhibition by vitamin B₁₂.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Noble gas supported boron-pentagonal clusters B<Subscript>5</Subscript>Ng<Subscript>n</Subscript><Superscript>3+</Superscript>: exploring the structures and bonding

A novel type of trivalent BNg five-membered cational species B₅Ng_n³⁺(Ng = He~Rn, n = 1~5) has been found and investigated theoretically using the B3LYP and MP2 methods with the def2-QZVPPD and def2-TZVPPD basis sets. The geometry, harmonic vibrational frequencies, bond energies, charge distribution, bond nature, aromaticity, and energy decomposition analysis of these structures were reported. The calculated B?Ng bond energy is quite large (the averaged bond energy is in the range of 209.2~585.76 kJ mol^-1) for heavy rare gases and increases with the Ng atomic number. The analyses of the molecular wavefunction show that in the BNg compounds of heavy Ng atoms Ar~Rn, the B?Ng bonds are of typical covalent character. Nuclear independent chemical shifts display that both B₅³⁺ and B₅Ng_n³⁺(n=1~5) have obvious aromaticity. Energy decomposition analysis shows that these BNg compounds are mainly stabilized by the σ-donation from the Ng valence p orbital to the B₅³⁺ LUMO. These findings offer valuable clues toward the design and synthesis of new stable Ng-containing compounds.     

Probiotic lactic acid bacterium from <Emphasis Type="Italic">kanjika</Emphasis> as a potential source of vitamin B<Subscript>12</Subscript>: evidence from LC-MS,immunological and microbiological techniques

Vitamin B₁₂ was produced by probiotic Lactobacillus plantarum in submerged fermentation (96 h) with successive anaerobic and aerobic phases of 48 h each to give 13 ng vitamin B₁₂/g dry biomass. Sodium cyanide-mediated cell lysis, followed by benzyl alcohol/chloroform/water extraction, improved the release of intracellular vitamin B₁₂ for analysis. The presence of the K⁺ adduct of cyanocobalamin (m/z of 1394) was established using electron spray ionization–mass spectra; growth of a mutant of Escherichia coli in the presence of cyanocobalamin ascertained its bioavailability.     

<Superscript>1</Superscript>H-NMR metabonomics analysis of sera differentiates between mammary tumor-bearing mice and healthy controls

Global analysis of ¹H-NMR spectra of serum is an appealing approach for the rapid detection of cancer. To evaluate the usefulness of this method in distinguishing between mammary tumor-bearing mice and healthy controls, we conducted ¹H-NMR metabonomic analyses on serum samples obtained from the following: 10 mice inoculated with a highly-metastatic mammary carcinoma cell line, 10 mice inoculated with a “normally” metastatic mammary carcinoma cell line, and 10 healthy controls. Following standard spectral processing and subsequent data reduction, we applied unsupervised Principal Component Analysis (PCA) to determine if unique metabolic fingerprints for different categories of metastatic breast cancer in serum exist. The PCA method correctly separated sera of tumor-bearing mice from that of normal healthy controls, as shown using the scores plot which indicated that sera classes from tumor-bearing mice did not share multivariate space with that from healthy controls. In addition, this technique was capable of distinguishing between classes of varying metastatic ability in this system. Metabolites apparently responsible for separation between diseased and healthy mice include lactate, taurine, choline, and sugar moieties. Results of this study suggest that ¹H-NMR spectra of mouse serum analyzed using PCA statistical methods indicate separation of tumor-bearing mice from healthy normal controls, justifying further study of the use of ¹H-NMR metabonomics for cancer detection using serum.     

Plasma free amino acid kinetics in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) using a bolus injection of <Superscript>15</Superscript>N-labeled amino acids

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of ¹⁵N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R _d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R _d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R _d values ranged from 0.9 μmol 100 g⁻¹ h⁻¹ (lysine) to 22.1 μmol 100 g⁻¹ h⁻¹ (threonine) with most values falling between 2 and 6 μmol 100 g⁻¹ h⁻¹. There was a significant correlation between R _d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.     

The effect of cold-induced increased metabolic rate on the rate of <Superscript>13</Superscript>C and <Superscript>15</Superscript>N incorporation in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

Animals with high metabolic rates are believed to have high rates of carbon and nitrogen isotopic incorporation. We hypothesized that (1) chronic exposure to cold, and hence an increase in metabolic rate, would increase the rate of isotopic incorporation of both ¹³C and ¹⁵N into red blood cells; and (2) that the rate of isotopic incorporation into red blood cells would be allometrically related to body mass. Two groups of sparrows were chronically exposed to either 5 or 22°C and switched from a ¹³C-depleted C₃-plant diet to a more ¹³C-enriched C₄-plant one. We used respirometry to estimate the resting metabolic rate of birds exposed chronically to our two experimental temperatures. The allometric relationship between the rate of ¹³C incorporation into blood and body mass was determined from published data. The of birds at 5°C was 1.9 times higher than that of birds at 22°C. Chronic exposure to a low temperature did not have an effect on the rate of isotopic incorporation of ¹⁵N save for a very small effect on the incorporation of ¹³C. The isotopic incorporation rate of ¹³C was 1.5 times faster than that of ¹⁵N. The fractional rate of ¹³C incorporation into avian blood was allometrically related to body mass with an exponent similar to −1/4. We conclude that the relationship between metabolic rate and the rate of isotopic incorporation into an animal’s tissues is indirect. It is probably mediated by protein turnover and thus more complex than previous studies have assumed.     

Effects of growth and tissue type on the kinetics of <Superscript>13</Superscript>C and <Superscript>15</Superscript>N incorporation in a rapidly growing ectotherm

The use of stable isotopes to investigate animal diets, habitat use, and trophic level requires understanding the rate at which animals incorporate the ¹³C and ¹⁵N from their diets and the factors that determine the magnitude of the difference in isotopic composition between the animal’s diet and that of its tissues. We determined the contribution of growth and catabolic turnover to the rate of ¹³C and ¹⁵N incorporation into several tissues that can be sampled non-invasively (skin, scute, whole blood, red blood cells, and plasma solutes) in two age classes of a rapidly growing ectotherm (loggerhead turtles, Caretta caretta). We found significant differences in C and N incorporation rates and isotopic discrimination factors (Δ¹³C = δ¹³C_tissues − δ¹³C_diet and Δ¹⁵N = δ¹⁵N_tissues − δ¹⁵N_diet) among tissues and between age classes. Growth explained from 26 to 100% of the total rate of incorporation in hatchling turtles and from 15 to 52% of the total rate of incorporation in juvenile turtles. Because growth contributed significantly to the rate of isotopic incorporation, variation in rates among tissues was lower than reported in previous studies. The contribution of growth can homogenize the rate of isotopic incorporation and limit the application of stable isotopes to identify dietary changes at contrasting time scales and to determine the timing of diet shifts. The isotopic discrimination factor of nitrogen ranged from −0.64 to 1.77‰ in the turtles’ tissues. These values are lower than the commonly assumed average 3.4‰ discrimination factors reported for whole body and muscle isotopic analyses. The increasing reliance on non-invasive and non-destructive sampling in animal isotopic ecology requires that we recognize and understand why different tissues differ in isotopic discrimination factors.     

A newly isolated and identified vitamin B<Subscript>12</Subscript> producing strain: <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 320

Vitamin B₁₂ (Cobalamin, VB₁₂) has several physiological functions and is widely used in pharmaceutical and food industries. A new unicellular species was extracted from China farmland, and the strain could produce VB₁₂ which was identified by HPLC and HPLC–MS/MS. 16S rDNA analysis reveals this strain belongs to the species Sinorhizobium meliloti and we named it S. meliloti 320. Its whole genome information indicates that this strain has a complete VB₁₂ synthetic pathway, which paves the way for further metabolic engineering studies. The optimal carbon and nitrogen sources are sucrose and corn steep liquor (CSL) plus peptone. The optimal combination of sucrose and CSL was obtained by response surface methodology as they are the most suitable carbon and nitrogen sources, respectively. This strain could produce 140 ± 4.2 mg L^?1 vitamin B₁₂ after incubating for 7 days in the optimal medium.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Theoretical study of the novel sandwich compound [Au<Subscript>3</Subscript>Cl<Subscript>3</Subscript>Tr<Subscript>2</Subscript>]<Superscript>2+</Superscript>

A theoretical study of a sandwich compound with a metal monolayer sheet between two aromatic ligands is presented. A full geometry optimization of the [Au₃Cl₃Tr₂]²⁺ (1) compound, which is a triangular gold(I) monolayer sheet capped by chlorines and bounded to two cycloheptatrienyl (Tr) ligands was carried out using perturbation theory at the MP2 computational level and DFT. Compound (1) is in agreement with the 18–electron rule, the bonding nature in the complex may be interpreted from the donation interaction coming from the Tr rings to the Au array, and from the back-donation from the latter to the former. NICS calculations show a strong aromatic character in the gold monolayer sheet and Tr ligands; calculations done with HOMA, also report the same aromatic behavior on the cycloheptatrienyl fragments giving us an insight on the stability of (1). The Au –Au bond lengths indicate that an intramolecular aurophilic interaction among the Au(I) cations plays an important role in the bonding of the central metal sheet. Figure (a) Ground state geometry of complex 1; (b) Top view of compound 1 and Wiberg bond orders computed with the MP2/B1 computational method; (c) Lateral view of compound 1 and NICS values calculated with the MP2/B1 method; the values in parenthesis were obtained at the VWN/TZP level     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

Determination of Oxidative Glucose Metabolism In Vivo in the Young Rat Brain Using Localized Direct-Detected <Superscript>13</Superscript>C NMR Spectroscopy

Determination of oxidative metabolism in the brain using in vivo ¹³C NMR spectroscopy (¹³C MRS) typically requires repeated blood sampling throughout the study to measure blood glucose concentration and fractional enrichment (input function). However, drawing blood from small animals, such as young rats, placed deep inside the magnet is technically difficult due to their small total blood volume. In the present study, a custom-built animal holder enabled temporary removal of the animal from the magnet for blood collection, followed by accurate repositioning in the exact presampling position without degradation of B₀ shimming. ¹³C label incorporation into glutamate C4 and C3 positions during a 120 min [1,6-¹³C₂] glucose infusion was determined in 28-day-old rats (n = 4) under α-chloralose sedation using localized, direct-detected in vivo ¹³C MRS at 9.4T. The tricarboxylic acid cycle activity rate (V _TCA) determined using a one-compartment metabolic modeling was 0.67 ± 0.13 μmol/g/min, a value comparable to previous ex vivo studies. This methodology opens the avenue for in vivo measurements of brain metabolic rates using ¹³C MRS in small animals.     

Pressure dependence of side chain <Superscript>13</Superscript>C chemical shifts in model peptides Ac-Gly-Gly-Xxx-Ala-NH<Subscript>2</Subscript>

For evaluating the pressure responses of folded as well as intrinsically unfolded proteins detectable by NMR spectroscopy the availability of data from well-defined model systems is indispensable. In this work we report the pressure dependence of ¹³C chemical shifts of the side chain atoms in the protected tetrapeptides Ac-Gly-Gly-Xxx-Ala-NH₂ (Xxx, one of the 20 canonical amino acids). Contrary to expectation the chemical shifts of a number of nuclei have a nonlinear dependence on pressure in the range from 0.1 to 200 MPa. The size of the polynomial pressure coefficients B ₁ and B ₂ is dependent on the type of atom and amino acid studied. For H^N, N and C^α the first order pressure coefficient B ₁ is also correlated to the chemical shift at atmospheric pressure. The first and second order pressure coefficients of a given type of carbon atom show significant linear correlations suggesting that the NMR observable pressure effects in the different amino acids have at least partly the same physical cause. In line with this observation the magnitude of the second order coefficients of nuclei being direct neighbors in the chemical structure also are weakly correlated. The downfield shifts of the methyl resonances suggest that gauche conformers of the side chains are not preferred with pressure. The valine and leucine methyl groups in the model peptides were assigned using stereospecifically ¹³C enriched amino acids with the pro-R carbons downfield shifted relative to the pro-S carbons.     

Novel vitamin B<Subscript>12</Subscript>-producing <Emphasis Type="Italic">Enterococcus</Emphasis> spp. and preliminary in vitro evaluation of probiotic potentials

Vitamin B₁₂ is an essential nutrient required for crucial metabolic processes in humans. Vitamin B₁₂-producing lactic acid bacteria (LAB) have been attracting increased attentions currently because of the generally recognized as safe (GRAS) status. Most of recent studies focused on Lactobacillus, and little is known about B₁₂-producing Enterococcus. In the present study, five Enterococcus strains isolated from infant feces were identified as vitamin B₁₂ producers. Among them, Enterococcus faecium LZ86 had the highest B₁₂ production (499.8 ± 83.7 μg/L), and the B₁₂ compound from LZ86 was identified as the biological active adenosylcobalamin, using reversed phase high-performance liquid (RP-HPLC) chromatogram. We examined basic probiotic and safety properties of E. faecium LZ86 and found that it was able to survive harsh environmental conditions (hot temperature, cold temperature, ethanol and osmotic stresses), tolerate gastric acid (pH 2.0, 3 h) and bile salts (0.3%), and adhere to Caco-2 cells. We also showed that E. faecium LZ86 is devoid of transferable antibiotic resistance and potential virulence factors. Together, here we report a B₁₂-producing E. faecium strain LZ86 firstly, which has desirable probiotic properties and may serve as a good candidate for vitamin B₁₂ fortification in food industry.