Structural characterization of pertussis toxin A subunit

The relationship between the structure of the A subunit of pertussis toxin and its function was analyzed. Limited tryptic digestion of the A subunit converted the protein to two stable fragments (Mr = 20,000 and 18,000). Antibodies raised to synthetic peptides homologous to regions in the A subunit were used to map these fragments. Both fragments were shown to contain the NH2-terminal portion but not the COOH-terminal portion of the A subunit. While these fragments exhibited NAD glycohydrolase activity, they were unable to reassociate with the B oligomer of the toxin. Thus the COOH-terminal portion of the A subunit does not contain the residues which are required for the NAD glycohydrolase activity of the toxin. However, this region of the molecule may be important for maintaining the oligomeric structure of the toxin. These results suggest that the A subunit of pertussis toxin is similar in structure to the A subunit of cholera toxin. In addition, antibodies raised to a synthetic peptide identical to residues 6-17 of the A subunit of pertussis toxin will bind to the A subunit of cholera toxin.     

Expression of CCAAT/Enhancer Binding Proteins during Porcine Preadipocyte Differentiation

The expression of three CCAAT/enhancer-binding proteins (C/EBPs) was examined with immunocytochemistry and Western blot analysis during preadipocyte differentiation in porcine stromal vascular (S-V) cell cultures. Regardless of treatment and time in culture, immunoreactivity for all three C/EBP isoforms was restricted to cell nuclei. At day 1, 50 ± 6% of S-V cells were C/EBPδ positive, whereas 13 ± 3 and 11.7 ± 3% of S-V cells were AD-3 and C/EBPα positive, respectively. After 3 days of seeding in fetal bovine serum (FBS) and dexamethasone (DEX), C/EBPδ; AD-3, and C/EBPα-positive cells increased to 67 ± 5, 42 ± 4, and 32 ± 3%, respectively. Double staining clearly showed that most of the C/EBPα reactive cells had not accumulated appreciable lipid after 3 days of FBS + DEX. Following 3 days of insulin treatment, the percentage of C/EBPδ cells was 50 ± 6, whereas the percentage of AD-3- and C/EBPα-positive cells was 41 ± 4 and 31 ± 3, respectively. After insulin treatment all fat cells were AD-3, C/EBPα, and C/EBPδ positive. Double staining demonstrated that fat cells were C/EBPδ reactive throughout the culture period. Western blotting showed changes in C/EBP isoform expression that were consistent with the immunocytochemical results. We conclude that C/EBPα is a terminal differentiation marker which is expressed later than AD-3 but further studies are needed to determine the relationship between C/EBPδ and adipogenesis in porcine S-V cultures.     

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Influence of Thyroxine In Vivo on Preadipocyte Development and Insulin-Like Growth Factor-I and IGF Binding Protein Secretion in Fetal Stromal Vascular Cell Cultures

Studies were conducted to determine the influence of thyroxine (T₄) in vivo on preadipocyte development and insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) secretion in stromal-vas-cular (S-V) cultures. Fetal pigs were hypophysectomized (hypox) at 70 days of gestation, implanted with T₄ pellets, and fetuses from the dam at 75 days of gestation. In a second experiment, hypox and T₄ implantation were performed at 75 days and fetal pigs removed at 95 days of gestation. Primary cultures of stromal vascular (S-V) cells derived from fetal adipose tissue were established. Cultures were stained for morphological analysis and conditioned media were collected for IGF-I determination by radioimmunoassay (RIA) and IGFBP analysis by Western blotting. After only 5 days of T₄ treatment, fat cell cluster number and size and lipid deposition in cultures were significantly increased compared to cultures from untreated hypox fetuses. Fetal hypox reduced IGF-I secretion by preadipocytes at both ages and T₄ treatment completely normalized IGF-I secretion (p < 0.05). Four IGFBPs (BP-1, BP-2, BP-3 & BP-4) detected in S-V cultures derived from 95-day fetuses were decreased in concentration by hypox by 44 ± 9.4%, 32 ± 9.7%, 42 ± 12% and 53 ± 6.9%. In cultures derived from T₄ treated hypox fetuses, the levels of these four IGFBPs were increased by 187 ± 25%, 239 ± 38%, 190 ± 5% and 347 ± 43% over control values, respectively. In cultures from 75-day fetuses, only IGFBP-2 (major one) and BP-1 (minor one) were detected and their secretion was also decreased by hypox and elevated by T₄ treatment (190 ± 49.5%, 156 ± 30%, respectively, of controls). The results provide direct evidence that T₄ has a major influence on fetal preadipocyte development. T₄ stimulated production of IGF-I and IGFBP in fetal S-V cultures, which in turn, may have mediated the capability of T₄ to enhance fetal adipose tissue development.     

Binding of ATP by pertussis toxin and isolated toxin subunits.

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of [3H]ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP greater than ATP greater than GTP greater than CTP greater than TTP for pertussis toxin and ATP greater than GTP greater than TTP greater than CTP for the B oligomer. Phosphate ions inhibited the binding of [3H]ATP to pertussis toxin in a competitive manner; however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of [3H]ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.     

Decreases in local hormone biosynthesis and c-fos gene expression accompany differentiation of porcine preadipocytes

Summary To better understand possible autocrine or paracrine mechanisms involved in adipose tissue development, we have studied the biosynthesis of insulinlike growth factor I (IGF-I) and prostaglandin E₂ (PGE₂) by cultured porcine preadipocytes in response to factors known to modulate cell growth and differentiation. The expression of c-fos was also monitored because of the potential role of that proto-oncogene in coordination of growth and differentiation. Preadipocytes were grown to confluence and then maintained in one of three media treatments: a) standard medium supplemented with 10% fetal bovine serum (FBS), b) FBS supplemented with dexamethasone (Dex), c) FBS supplemented with dibutryladenosine 3′–5′-cyclic monophosphate. Indirect measurements of growth indicated that cell proliferation did not differ due to media type. Histochemical and enzymatic measurements of adipocyte development revealed that differentiation occurred only in those cultures exposed to Dex. The increase in adipocyte differentiation in response to Dex was associated with a decrease in c-fos and actin RNA expression whereas the decrease in c-fos RNA expression in response to Dex was small (approximately 40%); immunocytochemical analysis indicated that induction of Fos protein occurred only in undifferentiated cells. Thus, the cells responsible for the decrease in c-fos RNA expression are possibly those signaled to differentiate into adipocytes. Expression of IGF-I RNA and secretion of IGF-I and PGE₂ were also decreased in response to Dex treatment. These data provide the first demonstration that biosynthesis of IGF-I by preadipocytes can be modulated by a potent inducer of adipocyte differentiation. The combined results indicate that glucocorticoids may stimulate adipocyte differentiation by suppressing intracellular and putative intercellular mitogenic signals. This work was supported in part by grant HD 18447 from the National Institutes of Health, Bethesda, MD (G. J. H.). Mention of a trade mark, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture or University of Georgia and does not imply its approval to the exclusion of other products that may be suitable.     

The histochemistry of developing adipocytes in primary stromal-vascular cultures of rat adipose tissue

Summary Histochemical analysis for NADP-dependent dehydrogenases, succinate dehydrogenase, NADH and NADPH-tetrazoleum reductases and esterase was conducted on primary cultures of adipose tissue stromal-vascular cells. Enzyme activities were restricted to clusters of lipid laden cells (adipocytes). The number of enzyme reactive adipocytes increased with length of culture. Coverslips were partially coated with collagen to allow comparisons of cell differentiation on coated (C-glass) and uncoated glass (U-glass) surface. There were no reactions for NADH- and NADPH-tetrazoleum reductases (TR) in cells on C-glass whereas adipocytes and stromal cells on U-glass were reactive. Glucose-6-phosphate (G6PDH) and 6-phosphogluconate (6PGDH) dehydrogenase activities were markedly demonstrated in both stromal cells and adipocytes on U-glass. Malate (MDH) and isocitrate (ICDH) dehydrogenase activites were higher in adipocytes than in stromal cells on the U-glass. Stromal cells on C-glass were either devoid of these enzymes (G6PDH, MDH, 6PGDH, ICDH) or activity was restricted to a small area of the cytoplasm. There were two levels of staining intensity in (MDH, ICDH, G6PDH, 6PGDH) adipocyte clusters on C-glass.Elimination of phenazine methosulphate from the NADP-dependent dehydrogeanse medias and SDH media, caused a reduction in enzyme reactive adipocytes on the C-glass. This manipulation did not reduce the number of enzyme reactive cells on U-glass. Cells on C-glass and U-glass were distinctly different in esterase stained coverslips. These studies demonstrated enzyme histochemical reactions of adipocytes and stromal cells in primary culture that were dependent on the type of extracellular matrix. Furthermore, enzyme histochemistry was shown to be useful for delineating adipocytes from stromal cells in primary cultures.