Influence of fat source and sorghum grain treatment on performance and digestibilities of high yielding dairy cows

Forty-eight lactating Holstein cows averaging 81 d in milk were allotted to eight blocks based on milk yield during the 14-d pretreatment period and randomly assigned to six treatment groups in a 2 x 3 factorial arrangement of treatments for 64 d. Factors were type of sorghum grain processing [dry-rolled vs. steam-flaked; fed at 34% of dry matter (DM) in a total mixed ration (TMR) based on alfalfa] and type of supplemental fat (2.5% of DM as cottonseed oil, tallow, or prilled fatty acids). Compared with dry-rolled sorghum, steam-flaked sorghum did not affect milk yield, fat percentage, or fat yield but did increase milk protein percentage, body weight gains, and estimated net energy for lactation (22%). Fat source did not affect lactational response, but, compared with tallow, prilled fatty acids tended to decrease DM intake. Steam-flaked sorghum, compared with dry-rolled sorghum, increased digestibilities of DM, organic matter, crude protein, and starch, regardless of fat source. The TMR containing prilled fat had lower digestibilities of DM and organic matter than did TMR containing cottonseed oil or tallow; and TMR containing prilled fat had lower digestibilities of crude protein and total fatty acids than did TMR containing tallow. This study showed that steam-flaking of sorghum grain increased milk protein content, body weight gains, and estimated net energy for lactation, regardless of dietary fat source.     

Method for measuring base/neutral and carbamate pesticides in personal dietary samples

Dietary uptake may be a significant pathway of exposure to contaminants. As such, dietary exposure assessments should be considered an important part of the total exposure assessment process. The objective of this work was to develop reliable methods that are applicable to a wide range of base/neutral and carbamate-type pesticides in duplicate diet samples collected as part of dietary exposure assessment studies. The resulting method needed to be sensitive to concentrations below 1 ng/g, accurate and precise, and as simple and cost effective as possible. As a first step, information was gathered on current methods for measuring pesticides in foods. Although the literature methods could serve as a starting point, few had been applied to duplicate diet samples and detection limits were generally high (10 to 100 ng/g). Experimental work was performed to evaluate individual extraction, cleanup, and analysis procedures; link the most promising procedures into analysis methods; and generate performance data on the final method. The final method used Soxhlet extraction with solvent partitioning and gel permeation chromatography cleanup. Gas chromatography/mass spectrometry was used for the analysis of base/neutral pesticides. High performance liquid chromatography analysis was used for the analysis of carbamate pesticides. Results of performance testing showed good accuracy (recovery > 70%), precision (% RSD < 25%), and sensitivity (method detection limits < 1.0 ng/g) for most pesticides targeted for study.     

Dynamic compatibility testing of DMP 840, an experimental antitumor agent

The purpose of this study is to evaluate the potential for DMP 840, a novel experimental antitumor agent, to precipitate during injection or dilution with infusion solutions. The influence of predilution of the drug solution before injection and addition of buffers to the drug vehicle were also investigated. The compatibility of normal saline solution, pH 7.4 phosphate buffers, and human plasma with DMP 840 was examined in vitro under both static conditions and dynamic flow. The combination of DMP 840 solutions with normal saline solution resulted in conversion of the drug to an insoluble dihydrochloride salt. Under conditions of dynamic flow, precipitation, accompanied by large changes in turbidity, occurred at relatively high concentrations of the drug in the injection solution. Dilution of the injection solution below 2 mg/mL or slow injection avoided precipitation. As was the case with the normal saline system, turbidity changes after injection into protein-phosphate buffer (PPB) were dependent on the initial concentration of DMP 840 solution as well as the rate of administration. In addition, the maximum injection rate at which complete miscibility occurred increased exponentially as the drug injection solution was made more dilute. Buffering the DMP 840 injection solution with acetate buffer improved the miscibility of DMP 840 with PPB, which indicated that the turbidity increases were most likely due to conversion of the drug to its insoluble free base form. The observed effects of the buffer on the turbidity response agreed qualitatively with predictions from a graphical approach that considers the effects of dilution and pH changes on drug solubility. Despite these observations, no evidence for the formation of a solid precipitate could be found after injection of the unbuffered drug solution into PPB. Further investigation indicated that the presence of albumin in the PPB prevented the formation of a solid phase during injection. Likewise, fresh human plasma, spiked with 1 and 2 mg/mL solutions of DMP 840, showed no evidence for the formation of a solid precipitate.     

Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum deltaH

Methanobacterium thermoautotrophicum deltaH was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F420 (factor F390-A) and other methanogenic cofactors. In addition, cells collected from the chemostat were measured for methyl coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F420-dependent and H2-dependent methylenetetrahydromethanopterin dehydrogenase (F420-MDH and H2-MDH, respectively), total (viologen-reducing) and coenzyme F420-reducing hydrogenase (FRH), factor F390 synthetase, and factor F390 hydrolase. The experiments were performed to investigate how the intracellular F390 concentrations changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F390 varied in a way that is consistently understood from the biochemical mechanisms underlying its synthesis and degradation. Moreover, a remarkable correlation was observed between expression levels of MCR I and II, F420-MDH, and H2-MDH and the cellular contents of the factor. These results suggest that factor F390 is a reporter compound for hydrogen limitation and may act as a response regulator of methanogenic metabolism.     

Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha

A human MSH2-human MSH3 (hMSH2.hMSH3) complex of approximately 1:1 stoichiometry (human MutSbeta (hMutSbeta)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSbeta supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSalpha (hMutSalpha), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSbeta. Thus, the repair specificities of hMutSalpha and hMutSbeta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSalpha level in repair-proficient HeLa cells (1.5 microg/mg nuclear extract) is approximately 10 times that of hMutSbeta. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSalpha, the hMutSbeta level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSalpha and hMutSbeta, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.     

Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli

An advantage of exporting a recombinant protein to the periplasm of Escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm. However, protein degradation in the periplasm also occurs. It has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli. To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) fused to alkaline phosphatase leader peptide was used as a model protein. A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm. It was found that the half-life of the barnase variants in vivo increased with their thermodynamic stability in vitro. A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant. The yield of a stabilized mutant was up to 50% higher than that of the wild type. This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.     

Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.