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Biomedical subjects

D C James

Publications and source records attributed to D C James.

At least 37 records · Page 2Linked to original sources
PubMed1998

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells.

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.

Animals↗
PubMed1998

Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period.

For a period of time after unilateral brain injury, surviving neural tissue surrounding the lesion may be vulnerable to extremely high behavioral demand. Previously, we found that lesions of the forelimb representation area of the sensorimotor cortex (FL-SMC) in rats increase in size substantially when the intact forelimb is immobilized with a plaster of paris cast during the first 15 days after surgery, which forces overuse of the impaired forelimb. The present study was designed to determine whether the adult brain is more vulnerable to forced overuse of the impaired forelimb during the first 7 days post-lesion than during the second 7 days post-lesion. Using behavioral tests of forelimb use and stereological analysis of remaining tissue volume 40 days after FL-SMC lesions, we found that forced overuse of the impaired forelimb during the first 7 days after the initial damage caused expansion of neural injury and greatly interfered with restoration of function. In contrast, forced overuse of the impaired forelimb during the second 7 days had no significant effect on lesion size but nevertheless interfered with restoration of function. Thus, surviving neural tissue in the damaged hemisphere and recovery of function appear to be vulnerable to prolonged forced overuse of the impaired forelimb throughout the first 15 days, but tissue loss was detectable only when the animal was forced to use the impaired forelimb during the first 7 days after injury.

Animals↗
PubMed1998

Curriculum integration in nutrition and mathematics.

Today's school-aged children face a multitude of health issues that affect their well-being and academic performance. Partnerships have developed between health and education agencies to help American children succeed at math and science and to prepare them to make healthful, lifelong decisions. Curriculum integration provides a framework for children to apply knowledge from several disciplines and to use this knowledge to solve real-life problems at work and at play. Goals for instruction focus on the needs not only of the individual but also of society. Nutrition science and mathematics form a natural partnership. Nutrition science incorporates numerous mathematical concepts and procedures such as sorting, classifying, statistics, probability, estimation, and rates and proportion. In preparation for participation in a global and technological society that will require citizens to be quantitative thinkers, educators must endeavor to assist all children in becoming adults who are mathematically literate and competent.

Adolescent↗
PubMed1997

Monitoring proteolysis of recombinant human interferon-gamma during batch culture of Chinese hamster ovary cells.

Proteolytic cleavage of recombinant human interferon-gamma (IFN-gamma) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN-gamma was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN-gamma polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN-gamma polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN-gamma polypeptide with "ragged" C-termini.

Amino Acid Sequence↗
PubMed1997

Coping with a new society: the unique psychosocial problems of immigrant youth.

When immigrant children and adolescents leave their country of origin to live in the United States, they leave behind a familiar language, culture, community, and social system. They also experience a variety of emotional and cognitive adjustments to the realities of life in the United States. Many of these conflicts and adjustments place immigrant children at increased risk for psychosocial problems, school failure, drug use, and other risk-taking behavior. Early identification of immigrant children at risk for these problems can help school personnel and health care providers plan culturally appropriate and effective interventions. This article discusses the potential psychosocial problems encountered by immigrant children in adjusting to a new home, school, and society, and offers suggestions for action.

Acculturation↗
PubMed1997

Using focus groups to develop a nutrition education video for high school students.

Focus group interviews were used to develop a nutrition education video and a teacher's guide for use in Florida high schools. Authors conducted a pilot and four focus group interviews with ninth grade students in five geographically distinct regions of Florida. Most students agreed that a video with scenarios or success stories would work well. Teens expressed interest in 10 topics: eating disorders; consequences of unhealthy eating; preparing quick, healthy meals; what constitutes a balanced diet; nutrition and fitness; weight control; food and the environment; the food guide pyramid; nutrition facts and fallacies; and food labels. Students suggested no more than three or four topics should be covered in the video, and the video cast should consist mainly of teen-agers with different body sizes, who were average, attractive, and from different ethnic backgrounds. Music was recommended only for transitions between scenes, as background, or during the credits.

Adolescent↗
PubMed1996

Use-dependent exaggeration of neuronal injury after unilateral sensorimotor cortex lesions.

Unilateral injury to the forelimb representation area of the sensorimotor cortex (FL-SMC) in adult rats causes over-reliance on the unimpaired forelimb for postural-motor movements, as well as overgrowth of layer V pyramidal cell dendrites in the homotopic cortex of the noninjured hemisphere. The overgrowth appears to be use-dependent because it can be prevented by restricting movements of the unimpaired forelimb. Additionally, restricting the unimpaired forelimb in animals with FL-SMC damage results in significantly greater behavioral dysfunction when examined 2 d after cast removal (compared to that after impaired-limb immobilization, or no limb immobilization). In the present study, the long-term behavioral and anatomical effects of limb immobilization were examined. Animals with FL-SMC lesions were fitted with casts immediately after the lesion that immobilized the impaired forelimb, the unimpaired forelimb, or neither forelimb for 15 d. Immobilization of the nonimpaired forelimb resulted in chronic prevention of dendritic growth and severe and chronic behavioral deficits. In addition, immobilization of the nonimpaired forelimb resulted in a dramatic exaggeration of the neuronal injury, presumably attributable to forced overuse of the impaired limb. Immobilization of the impaired forelimb resulted in no detectable neural changes and in only slightly increased and longer-lasting behavioral asymmetries (compared to nonimmobilized, lesioned animals), presumably attributable to mild disuse of the impaired limb. Immobilization of a single forelimb in nonlesioned rats resulted in no significant behavioral or anatomical changes. Together, these results suggest that although behavioral experience can enhance neural growth after brain injury, the region surrounding the injury may be vulnerable to behavioral pressure during the early postlesion period.

Animals↗
PubMed1996

Posttranslational processing of recombinant human interferon-gamma in animal expression systems.

We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodopera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.

Amino Acid Sequence↗
PubMed1996

Getting the glycosylation right: implications for the biotechnology industry.

Glycosylation is the most extensive of all the posttranslational modifications, and has important functions in the secretion, antigenicity and clearance of glycoproteins. In recent years major advances have been made in the cloning of glycosyltransferase enzymes, in understanding the varied biological functions of carbohydrates, and in the accurate analysis of glycoprotein heterogeneity. In this review we discuss the impact of these advances on the choice of a recombinant host cell line, in optimizing cell culture processes, and in choosing the appropriate level of glycosylation analysis for each stage of product development.

Animals↗
PubMed1996

Using focus group interviews to understand school meal choices.

Focus groups were used to examine adolescents' participation in school nutrition programs, their opinions about the meals served in these programs, and their suggestions for improving the meals. A pilot and four focus group interviews were conducted with ninth grade students in five geographically distinct regions of Florida. Male students and those from rural areas were more likely to participate in school meal programs than were females and those from suburban and urban areas. Students expressed intense dislike and distrust of school meals. School meals were criticized for taste, appearance, small serving sizes, and cost. Students overwhelmingly favored commercial food vendors serving lunch at school. Suggestions for improving school meals included improving the taste and appearance of food, offering a wider variety of foods, serving more fresh fruit and vegetables, lowering the price, increasing the serving sizes, teaching food service workers how to cook, and offering more condiments.

Adolescent↗
PubMed1995

N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems.

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.

Animals↗
PubMed1995

Roles of physicians, genetic counselors, and nurses in the genetic counseling process.

A self-administered questionnaire was mailed to 325 genetic counseling professionals in the southeastern United States and 204 (63%) completed it. Among those responding, 32% held master's degrees in genetic counseling, 30% M.D. degrees, 23% degrees in nursing, and 3% master's degrees in social work. While genetic counseling typically involves team effort, members were generally unclear regarding their roles in the process. Respondents rarely referred their clients to social workers but believe they should. Most did not believe that master's trained genetic counselors should be the only health professional who provide counseling. The respondents strongly supported goals that dealt with the educative aspect of counseling and protection of patient autonomy.

Academic Medical Centers↗
PubMed1994

High-resolution separation of recombinant human interferon-gamma glycoforms by micellar electrokinetic capillary chromatography.

Recombinant human interferon-gamma (IFN-gamma) glycoform populations produced by Chinese hamster ovary cells have been resolved by micellar electrokinetic capillary chromatography (MECC). Separations were performed in uncoated fused silica capillaries at alkaline pH in the presence of micellar concentrations of the anionic detergent sodium dodecyl sulfate (SDS). Maximum resolution was obtained reproducibly with high-ionic-strength borate/SDS electrophoresis buffer. Under the conditions described, glycoform migration time was inversely related to the amount of carbohydrate associated with the protein. Digestion of IFN-gamma with peptide-N-glycosidase F allowed virtual real-time monitoring of glycosidase digests by capillary electrophoresis. Analysis of other digestions with either neuraminidase or endoglycosidase H (endo H) showed most IFN-gamma glycoforms to be sialylated and a minor proportion of glycoforms to be associated with oligomannose structures. While both bovine pancreas ribonuclease B and horse-radish peroxidase glycoforms were separated by this technique, proteins glycosylated at multiple sites such as bovine serum fetuin and human alpha 1-acid glycoprotein were not well resolved by MECC.

Amidohydrolases↗
PubMed1990

The use of enzyme immunoassay (EIA) and radiobinding assay to investigate the cross-reactivity of Klebsiella antigens and HLA B27 in ankylosing spondylitis patients and healthy controls.

The binding of rabbit anti-Klebsiella antibodies to tissue-typed lymphocytes obtained from 30 ankylosing spondylitis (AS) patients and 54 healthy subjects has been measured by an enzyme immunoassay method. HLA B27 positive lymphocytes obtained from either AS patients (t = 3.60; p less than 0.001) or healthy subjects (t = 3.77; p less than 0.001) were found to bind Klebsiella antibodies to a significantly greater extent than non-B27 lymphocytes obtained from healthy controls. Absorption studies demonstrated that HLA B27 positive lymphocytes absorbed out significantly more anti-Klebsiella antibodies than HLA B27-negative lymphocytes (t = 6.76; p less than 0.005). These results are compatible with cross-reactivity or molecular mimicry between HLA B27 and epitopes on some Gram-negative bacteria such as Klebsiella.

Antigens, Bacterial↗