Influence of Primatone RL supplementation on sialylation of recombinant human interferon-gamma produced by Chinese hamster ovary cell culture using serum-free media

Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn(25) and Asn(97)) of IFN-gamma with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-gamma by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.     

Ultrasound biomicroscopy for longitudinal studies of carotid plaque development in mice: validation with histological endpoints

Atherosclerosis is responsible for the death of thousands of Americans each year. The carotid constriction model of plaque development has recently been presented as a model for unstable plaque formation in mice. In this study we 1) validate ultrasound biomicroscopy (UBM) for the determination of carotid plaque size, percent stenosis, and plaque development in live animals, 2) determine the sensitivity of UBM in detecting changes in blood flow induced by carotid constriction and 3) test whether plaque formation can be predicted from blood flow parameters measured by UBM. Carotid plaques were induced by surgical constriction in Apo E−/− mice. Arteries were imaged bi-weekly by UBM, at which time PW-Doppler measurements of proximal blood flow, as well as plaque length and percent stenosis were determined. Histology was performed 9 weeks post surgery. When compared to whole mount post-mortem measurements, UBM accurately reported carotid plaque length. Percent stenosis, based on transverse B-mode UBM measurements, correlated well with that calculated from histological sections. PW-Doppler revealed that constriction reduced maximum systolic velocity (v_max) and duration of the systolic velocity peak (t_s/t_t). Pre-plaque (2 week post-surgery) PW-Doppler parameters (v_max and t_s/t_t) were correlated with plaque length at 9 weeks, and were predictive of plaque formation. Correlation of initiating PW-Doppler parameters (v_max and t_s/t_t) with resulting plaque length established the degree of flow disturbance required for subsequent plaque development and demonstrated its power for predicting plaque development.     

A spatial analytical approach for selecting reintroduction sites for burbot in English rivers

1. Availability of suitable habitat is a prerequisite for species reintroduction success, and to ensure population persistence, investigations of a species’ habitat utilisation throughout its life history should be conducted as part of a feasibility study. 2. Habitat utilisation models for burbot, Lota lota, developed using data from field studies conducted in France and Germany and information from the literature were used to assess the feasibility of reintroducing burbot into rivers of its former native range in eastern England. 3. Per cent tree roots, aquatic vegetation and flow types were important predictors of adult burbot abundance. Furthermore, the habitat utilisation models were supplemented with information from the literature, which suggested that off‐channel habitat such as wetlands and backwaters is important for spawning and nursery stages. 4. An assessment of the habitat availability in the rivers of the burbot’s former native range using variables related to spawning and nursery and adult life stages showed that although adult habitat was widely distributed, the availability of spawning and nursery habitat was less abundant, potentially limiting successful reestablishment. 5. Potential suitable habitat was concentrated in the central and southern areas of the species’ former English distribution. Overall, rivers of the burbot’s former range potentially afford suitable habitat to sustain a reintroduced population. However, sites should be preferentially selected on the basis of having appropriate spawning and nursery areas.     

Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide

Cytochrome P-450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular reactivity and function. Conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases is thought to be the major pathway of EET metabolism in mammalian vascular cells. However, when human coronary artery endothelial cells (HCEC) were incubated with (3)H-labeled 14,15-EET, chain-shortened epoxy fatty acids, rather than DHET, were the most abundant metabolites. After 4 h of incubation, 23% of the total radioactivity remaining in the medium was converted to 10,11-epoxy-hexadecadienoic acid (16:2), a product formed from 14,15-EET by two cycles of beta-oxidation, whereas only 15% was present as 14,15-DHET. Although abundantly present in the medium, 10,11-epoxy-16:2 was not detected in the cell lipids. Exogenously applied (3)H-labeled 10,11-epoxy-16:2 was neither metabolized nor retained in the cells, suggesting that 10,11-epoxy-16:2 is a major product of 14,15-EET metabolism in HCEC. 10,11-Epoxy-16:2 produced potent dilation in coronary microvessels. 10,11-Epoxy-16:2 also potently inhibited tumor necrosis factor-alpha-induced production of IL-8, a proinflammatory cytokine, by HCEC. These findings implicate beta-oxidation as a major pathway of 14,15-EET metabolism in HCEC and provide the first evidence that EET-derived chain-shortened epoxy fatty acids are biologically active.     

Mitigation of avian reproductive tract function by Salmonella enteritidis producing high-molecular-mass lipopolysaccharide

Hens were infected with a wild-type Salmonella enteritidis and its wzz mutant, which lacked the ability to make high-molecular-mass lipopolysaccharide (LPS), in six experiments paired by dosage and route of exposure. Involution of the reproductive tract occurred in 86% of hens that were injected subcutaneously with 108 cfu of the wild-type strain, but none did so when injected with the wzz mutant. In spite of the lack of a specific effect on the reproductive tract, infection of hens with the mutant produced more contaminated eggs and heterophilic granulomas in developing ova (yolks) than wild type; thus, overall, the mutant appeared to be more virulent except after intravenous injection. The mutant also decreased shell quality more often than wild type, regardless of dosage or route of infection. These results suggest that egg-contaminating Salmonella enteritidis that produces high-molecular-mass LPS mitigates signs of illness in poultry by altering the response of the avian reproductive tract to infection, but without altering the incidence of egg contamination following bacteraemia. Further research is warranted to determine whether analyses of shell quality might aid in identification of flocks at risk of producing contaminated eggs.     

Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes

Fatty acid translocase (FAT)/CD36 is one of several putative plasma membrane long-chain fatty acid (LCFA) transport proteins; however, its role in intestinal absorption of LCFA is unknown. We hypothesized that FAT/CD36 would be differentially expressed along the longitudinal axis of the gut and during intestinal development, suggesting specificity of function. We found that intestinal mucosal FAT/CD36 mRNA levels varied by anatomic location along the longitudinal gut axis: stomach 45 +/- 7, duodenum 173 +/- 29, jejunum 238 +/- 17, ileum 117 +/- 14, and colon 9 +/- 1% (means +/- SE with 18S mRNA as control). FAT/CD36 protein levels were also higher in proximal compared with distal intestinal mucosa. Mucosal FAT/CD36 mRNA was also regulated during intestinal maturation, with a fourfold increase from neonatal to adult animals. In addition, FAT/CD36 mRNA levels and enterocyte LCFA uptake were rapidly downregulated by intraduodenal oleate infusion. These findings suggest that FAT/CD36 plays a role in the uptake of LCFA by small intestinal enterocytes. This may have important implications in understanding fatty acid absorption in human physiological and pathophysiological conditions.