Culture nutrition and physiology impact the inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124.

Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis.

Nitrogen source and mineral optimization enhance D: -xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.

Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.

27-Hydroxycholesterol inhibits neutral sphingomyelinase in cultured human endothelial cells.

To study the effect of 27-hydroxycholesterol (27OHC) on the catabolism of sphingomyelin, we cultured endothelial cells (ECs) from human umbilical veins with 27OHC, then measured activities of acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) and sphingomyelin consumption by using [14C]sphingomyelin, and determined NSMase mRNA expressions by RT-PCR method. The results indicated that [14C]sphingomyelin accumulated in cells treated with 27OHC, and that the activities of both NSMase and ASMase were inhibited in ECs cultured with 27OHC. To further study the effect of 27OHC on NSMase, we used desipramine, an inhibitor of ASMase, to exclude the possible interference of ASMase's residual activity at neutral condition. Also, we observed the significant inhibition of NSMase activity by using glutathione, an inhibitor of NSMase, but found no further impact when 27OHC was added later. To determine whether the inhibition of NSMase activity was directly due to the effect of 27OHC, we exposed cell homogenate to 27OHC, and found no inhibitive effect of 27OHC on the activity of NSMase. All of our data confirmed that 27OHC had only an indirect inhibitive effect on NSMase. Our finding that no change of the NSMase mRNA expression by 27OHC indicated that the inhibitive effect of 27OHC on NSMase activity occurred at a post-transcriptional level. We suggest that an altered membrane fluidity caused by 27OHC could be involved in the inhibited activity of NSMase.

A 3800 gene microarray for cattle functional genomics: comparison of gene expression in spleen, placenta, and brain.

A cDNA microarray representing approximately 3800 cattle genes was created for functional genomic studies. The array elements were selected from > 7000 cDNA clones identified in a large-scale expressed sequence tag (EST) project that utilized spleen and normalized and subtracted placenta cDNA libraries. Sequence similarity searches of the 3820 ESTs represented on the array using BLASTN identified 3290 (86.1%) as putative human orthologs, with the remainder consisting of "novel" genes or highly divergent orthologs. Experiments were conducted with a prototype 768 gene microarray created from spleen cDNAs and with the 3800 gene array that included genes from spleen and placenta. The 768 gene array was used to profile RNA transcripts expressed by adult and fetal spleen. The 3800 gene array was used to profile transcripts expressed by adult brain and placenta. Microarray analysis of RNA extracted from fetal and adult spleen identified 29 genes that were differentially expressed two-fold or more. Transcriptional differences of two of these genes, IGJ and CTSS, were confirmed using TaqMan technology. The comparison of brain and placenta revealed 400 genes expressed at higher levels in brain and 72 genes expressed at higher levels in placenta. These results demonstrate the potential power of microarrays for understanding the molecular mechanisms of cattle development, disease resistance, nutrition, fertility and production traits.

Cloning and characterization of feline brain natriuretic peptide.

Brain (B-type) natriuretic peptide (BNP) is a cardiac hormone involved in regulation of fluid balance and blood pressure homeostasis of mammalian species. BNP sequence is species-specific and considered to be a significant prognostic and diagnostic marker for cardiac dysfunction. Using conventional polymerase chain reaction and amplification of cDNA 3'- and 5'-ends, a total of 1500 nucleotides encompassing the entire feline BNP gene were characterized. The feline BNP gene is organized in three exons separated by two introns. The complete transcript of 736 nucleotides was characterized, including 396 nucleotides encoding feline preproBNP. The preproBNP consisted of a signal peptide of 26 amino acids and a proBNP of 106 residues. The predicted mature BNP comprised 35 amino acids with likely 26- and 29-aa isomers, including a histidine residue at the C-terminus. Based on the similarity of BNP prepropeptide sequences, a phylogenetic relationship is presented for mammalian species including human, cat, cattle, dog, mouse, rat, sheep and swine.

Genomic sequence and cardiac expression of atrial natriuretic peptide in cats.

OBJECTIVE: To determine the nucleotide and amino acid sequence of atrial natriuretic peptide (ANP) in cats and its typical regions of cardiac expression. ANIMALS: 5 healthy adult mixed-breed cats. PROCEDURE: Total RNA was extracted from samples obtained from the left and right atrium, left and right ventricle, and interventricular septum of each cat. The RNA was used to produce cDNA for sequencing and northern blot analysis. Genomic DNA was extracted from feline blood samples. Polymerase chain reaction primers designed from consensus sequences of other species were used to clone and sequence the feline ANP gene. RESULTS: The feline ANP gene consists of 1,072 nucleotides. It consists of 3 exons (123, 327, and 12 nucleotides) separated by 2 introns (101 and 509 nucleotides). It has several typical features of eukaryotic genes and a putative steroid-response element located within the second intron. Preprohormone ANP consists of 153 amino acids. The amino acid sequence of the active form of feline ANP (ANP-30) is identical to that of equine, bovine, and ovine ANP-30 and differs from that of human, canine, and porcine ANP-28 only by 2 carboxy-terminal arginine residues. The ANP mRNA was detected only in the left and right atria. CONCLUSIONS AND CLINICAL RELEVANCE: The genetic and protein structure and principal regions of cardiac expression of feline ANP are similar to those of other species. Results of this study should be helpful in future studies on the natriuretic response in cats to diseases that affect cardiovascular function.

Monoclonal Antibody-Based ELISA for Detection of Clostridium perfringens Alpha-Toxin.

A monoclonal antibody-based ELISA was developed to detect alpha-toxin present in Clostridium perfringens bacterial cell lysates and cell-free culture supernatants, Monoclonal antibodies against C. perfringens alpha-toxin were produced in hybridoma tissue culture supernatants and in BALB/c mice ascites fluid, The monoclonal antibodies obtained from hybridoma culture supernatant and ascites fluid showed identical antigen specificity, but the latter showed a higher titer, with a 50% endpoint at 1/4,000. The monoclonal antibodies were specific for phospholipase C produced by C. perfringens , but not by Bacillus cereus . The lower limit of phospholipase C detection was 16 ng/ml. The dose-dependent relationship between absorption at 490 nm and concentration of phospholipase C diluted in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) or Trypticase glucose yeast broth fit a four-parameter and a quadratic model, respectively. The monoclonal antibody-based ELISA developed is a rapid, sensitive and specific detection method and can be used for quantitative characterization of C. perfringens alpha-toxin.