Comparison of the Ca2+ complexing properties of isosaccharinate and gluconate - is gluconate a reliable structural and functional model of isosaccharinate?

The calcium complexation and acid-base properties of α-d-isosaccharinate (Isa-) in neutral and in (hyper)alkaline solutions have been investigated via potentiometric titrations, multinuclear NMR, ESI-MS and quantum chemical calculations. Isa- is the primary alkaline degradation product of cellulose, and may be present in radioactive waste repositories and therefore, it could contribute to the mobilization of radioactive nuclei. Because of its limited availability, d-gluconate (Gluc-) is commonly used as a structural and functional model of Isa-. Therefore, the thermodynamic and structural data obtained for Isa- were compared with those of Gluc-. The formation constants of the CaIsa+ and CaGluc+ complexes present in neutral solutions are practically identical, but the binding sites are in different positions and the CaIsa20 solution species cannot be detected. The stepwise formation constant of the CaIsaH-10 complex (forming in alkaline medium) is somewhat larger than that of CaGlucH-10, which is in line with the observation that IsaH-12- is a stronger base than GlucH-12-. The most striking difference is that, unlike Gluc-, Isa- does not form polynuclear complexes with Ca2+. The structural reason for this is that the alcoholate groups on C2 and C3 adjacent to the carboxylate moiety on Gluc- are able to simultaneously bind Ca2+, making the formation of polynuclear Ca-complexes possible. On Isa-, only the alcoholate on C2 is involved, while the other one on C6 is not (supposedly for steric reasons). In conclusion, during the interactions of Gluc- and Isa- with Ca2+, differences rather than similarities prevail.

Estrogenic activity of musk fragrances detected by the E-screen assay using human mcf-7 cells.

The widespread use of synthetic musk fragrances and the resultant presence of these substances and their metabolites in the aquatic environment (as well as their accumulation in human adipose tissue) raises the question of whether musk fragrances display endocrine and in particular estrogenic activity. A variety of musk fragrances were tested using the E-screen assay. A statistically significant increase in proliferation rate of human MCF-7 breast cancer cells was detected for two nitro musks (musk xylene, musk ketone), a major metabolite of musk xylene ( p-amino-musk xylene), and the polycyclic musk fragrance AHTN. This indicates that these substances do, in fact, demonstrate estrogenic activity. Coincubation with the antiestrogen tamoxifen showed that the increase in proliferation rate by the musk fragrances is estrogen receptor-mediated. It must be noted, however, that the effective estrogenic strength and estrogenic potency were low compared to 17 b-estradiol. The naturally occurring fragrance muscone from the group of macrocyclic musk fragrances, a group of substances that have not yet been well characterized in respect to their toxicological properties, has also been shown to be weakly estrogenically active in vitro. E-screen analysis showed that the nitro musk metabolites o-amino musk xylene and 2-amino-MK, the macrocyclic musk fragrances ethylene brassylate, ethylene dodecandioate, and cyclopentadecanolide, are not estrogenically active.

Mutants of smooth muscle myosin light chain kinase at tryptophan 800.

The importance of Trp 800 in the calmodulin-binding site of myosin light chain kinase was investigated. Truncation mutants from Leu 447 to the C-terminus were expressed in E. coli and these were modified by point mutations of Trp 800 to Gly, Cys, Leu and Tyr. Trp at this position was more effective than any of the other residues. The Leu mutant was partially active and its Km for calmodulin decreased from about 10 nM to 175 nM. The Tyr mutant had detectable activity but the other two mutants were inactive and did not bind calmodulin. Thus Trp at position 800 is critical. The activity of the Leu mutant at high calmodulin concentrations was less than the wild-type mutant, about 20%. This suggests that the binding of calmodulin does not release inhibition in an all-or-none mechanism and that other intramolecular interactions are important.

Comparison of nitrogen-15 and diaminopimelic acid for estimating bacterial protein synthesis of lactating cows fed diets of varying protein degradability.

Three lactating Holstein cows fitted with duodenal cannulae were fed diets containing cottonseed meal, corn gluten meal, or blood meal as protein supplements in a 3 c 3 Latin square experiment. Diets averaged 15% CP and were 60% concentrate, 31% corn silage, and 9% alfalfa hay. The flow marker was Cr2O3; the bacterial protein fraction of digesta CP was estimated by 15N (as ammonium sulfate) and diaminopimelic acid. The undegraded fraction of total feed protein entering the duodenum for respective diets was .52, .57, and .69. The 15N method was less variable than diaminopimelic acid. Based on 15N, percentage of bacterial of total protein differed among treatments (61.5, 59.4, and 55.0, respectively). Ten percent more protein entered the duodenum on blood meal than other diets, but differences were not significant. Protein sources were similar in microbial passage, but degraded protein was used most efficiently for microbial synthesis on blood meal. Incorporation of 15N consumed into bacterial protein ranged from 50 to 83% with numerically highest values on blood meal, suggesting greater efficiency of ammonia, capture. Recoveries of 15N for the 72 h as milk, feces and urine ranged from 54 to 78%.

Influence of cultures of Aspergillus oryzae on rumen and total tract digestibility of dietary components.

Three trials were conducted to evaluate the effect of dried cultures of Aspergillus oryzae on nutrient utilization by mature Holstein cows fitted with ruminal and duodenal cannulas. In trial 1, four cows (two dry and two lactating) were used to test Aspergillus oryzae (3 g/d) and a control treatment at two forage amounts in a 4 x 4 Latin square. Trial 2 compared control, A. oryzae, and Saccharomyces cerevesiae using six lactating cows in a repeated 3 x 3 Latin square design. For trial 3, four lactating cows were used in a switchback design to compare control to A. oryzae treatment. In trials 2 and 3, diets contained 70 and 61% concentrate. A fourth in vitro trial was conducted in conjunction with trial 3 in which rumen fluid was obtained from cows adapted or unadapted to A. oryzae. On all trials except high forage in trial 1, A. oryzae increased rumen and total tract digestibility of fiber fractions. Rumen VFA and ammonia were not affected by fungal cultures. Aspergillus oryzae increased rate of rumen fermentation of alfalfa but not of milo or wheat straw. In vitro disappearance of DM from alfalfa, milo, and wheat straw was increased by A. oryzae, and previous adaptation was not required to stimulate in vitro DM digestibility. These results indicate that a primary effect of A. oryzae is stimulation of fiber digestion by rumen microbes.