Alternative pathways of zeaxanthin biosynthesis in a Flavobacterium species. Experiments with nicotine as inhibitor

In Flavobacterium R1519, nicotine blocks zeaxanthin biosynthesis by specifically inhibiting the cyclization reaction. Lycopene (at high nicotine concentrations, e.g. 7.5mm) and rubixanthin (at low nicotine concentration, e.g. 1mm) replace zeaxanthin as the main carotenoid. On removal of the nicotine lycopene is converted into beta-carotene under anaerobic conditions and into zeaxanthin in the presence of O(2). The conversion in vivo of beta-carotene into zeaxanthin was also demonstrated. Cyclization (an anaerobic process) thus precedes hydroxylation (O(2)-requiring) in the biosynthesis of zeaxanthin. The conversion in vivo of rubixanthin into beta-cryptoxanthin and into zeaxanthin was demonstrated, thus indicating the operation of alternative pathways of zeaxanthin biosynthesis. Several alternative biosynthetic pathways are considered and the results are also discussed in terms of reaction sequences of carotenoid ;half-molecules'.     

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed‐batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top‐down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1023–1033, 2013     

The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: effects of antimineralocorticoids

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.     

Pathway of inactivation of cholecystokinin octapeptide (CCK-8) by synaptosomal fractions

Cholecystokinin octapeptide (CCK-8) was degraded by peptidases present in intact synaptosomes isolated from rat cortex and hypothalamus. Most of the degrading activity was present in the cytoplasmic fraction although a small amount (7%) was membrane-bound. Products of the degradation were isolated by HPLC and characterized by amino acid analysis. The Met³-Gly⁴ bond was the main primary site of cleavage giving rise to Asp-Tyr(SO₃H)-Met and Gly-Trp-Met-Asp-Phe-NH₂. These products appeared to be further degraded by sequential removal of amino-terminal residues. The Asp¹-Tyr² and Asp⁷-Phe⁸ bonds were also sites of cleavage. p-Chloromercuribenzoate was the most effective inhibitor (90% inhibition) of CCK-8 degradation by synaptosomal peptidases at the concentrations tested.This peptidase activity in synaptosomes may be important in the regulation of levels of the neuropeptide CCK-8 at the synapse. Identification of the sites of cleavage of CCK-8 on incubation with synaptosomes will assist in the isolation and characterization of the enzymes involved.     

Maternal nest building by pseudopregnant mice.

Pseudopregnant mice displayed maternal nest-building behaviour. Their nests were smaller but qualitatively identical to those constructed by pregnant animals.