Steady-state response of quadriplegic subjects to inspiratory resistive load

Normal subjects preserve tidal volume (VT) in the face of added inspiratory resistance by increasing maximal amplitude and duration of the rising phase of respiratory driving pressure (DP) and by changing the shape of this phase to one that is more concave to the time axis. To explore the possible role of chest wall afferents in mediating these responses, we determined averaged DP in eight quadriplegic subjects during steady-state unloaded breathing and while breathing through an inspiratory resistance (8.5 cmH2O X 1(-1) X s). As with normal subjects, quadriplegics preserved VT (loaded VT = 106% control) by utilizing all three mechanisms. However, prolongation of the inspiratory duration derived from the DP waveform (+22% vs. +42%) and shape response were significantly less in the quadriplegic subjects. Shape response was completely absent in subjects with C4 lesions. The results provide strong evidence that respiratory muscle spindles are responsible for shape response and that changes in afferent feedback from the chest wall play an important role in mediating inspiratory prolongation.     

Selectivity in the binding of hydroxylated benzo[a]pyrene derivatives to purified cytochrome P-450c

The interaction of rat liver microsomal cytochrome P-450c with potential benzo[a]pyrene (BP) metabolites has been compared with the binding of BP by optical and fluorescence spectroscopy. Fluorescence quenching of the phenolic derivatives of BP derives from 1:1 complex formation with P-450c, is a function of the position of the hydroxyl substituent, and correlates with the concomitant increase in high-spin cytochrome observed in parallel optical titrations. The proportion of high-spin cytochrome seen when P-450c was reconstituted in dilauroylphosphatidylcholine vesicles (60 micrograms/mL) ranged from about 7% for the 3- and 7-phenols to 75% for 11- and 12-phenols. BP and all 12 methyl-BP derivatives have comparable high affinities for P-450c (50-70% high spin). Kd determinations with purified P-450c indicated very strong binding of BP phenols that induce high-spin complexes (4-, 5-, 9-, 10-, 11-, and 12-phenols; Kd = 3-25 nM). Inhibition of n-octylamine binding by the 3- and 7-phenols indicated weak interactions (Kd = 80-90 nM), even though low-spin complexes were formed. Inhibition of BP metabolism catalyzed by P-450c with BP phenols correlated with their respective dissociation constants. These results suggest that phenolic substitution at certain positions on BP (1, 2, 3, 7, or 8) interferes with binding to the active site while substitutions at the other positions either enhance or have no effect on binding. BP dihydrodiols [including the (+)- and (-)-BP 7,8-dihydrodiols] were relatively ineffective in forming high-spin complexes (approximately 20%), and fluorescence quenching of dihydrodiols by P-450c also saturated at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a novel ganglioside on erythrocytes with blood group Cad specificity

The blood group Cad antigen is a carbohydrate structure well characterized on the sialoglycoproteins of the red cell membrane from some rare individuals (Blanchard, D., Cartron, J. P., Fournet, B., Montreuil, J., Van Halbeck, H., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 7691-7695). However, protease treatment of whole cells did not destroy their antigenic activity which indicated that glycolipid might also be involved in the antigenic reaction. A crude ganglioside fraction was prepared from Cad cells and found to inhibit the hemagglutination reaction, whereas neutral glycolipids were inactive. Further analysis of the ganglioside extract from Cad erythrocytes by thin layer chromatography revealed an unusual profile characterized by a lower content of sialosylparagloboside and the presence of a novel ganglioside of slower mobility. Immunochemical studies demonstrate that this ganglioside binds Helix pomatia lectin and inhibits human anti-Sda antibody. In addition, a ganglioside with identical chromatographic mobility can be obtained by the enzymatic transfer of GalNAc from UDP-GalNAc to sialosylparagloboside using a microsomal preparation from human kidney. These results together with cell surface labeling experiments suggest that the major ganglioside of Cad erythrocytes might be derived from sialosylparagloboside by substitution with an additional N-acetylgalactosamine residue.     

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.     

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.     

Dysgenesis-Induced Instability of Rosy Locus Transformation in DROSOPHILA MELANOGASTER: Analysis of Excision Events and the Selective Recovery of Control Element Deletions

Utilizing the method of P-M hybrid dysgenesis-mediated gene transfer to insert rosy locus DNA into various chromosomal locations, we recovered a transformed strain that carries an ry+ transposon inserted in or near the scalloped locus in polytene section 13F on the X chromosome. The resultant product, when stabilized, behaves as a homozygous and hemizygous viable and fertile extreme scalloped allele associated with wild-type expression of the rosy locus. We have labeled this allele, sdry+. This allele has been destabilized by subsequent P-M hybrid dysgenesis, and mutations were recovered that exhibit alterations in the rosy and/or scalloped phenotypes. Representative samples of all phenotypic classes have been characterized by Southern blot analyses of restricted DNA. The most common events are excisions of DNA wholly internal to the transposon and representing sections of rosy DNA. In addition to loss of rosy locus function, such excisions affect the scalloped locus expression.--A second dysgenesis experiment was carried out involving an ry+ transposon inserted in polytene section 16D on the X chromosome. A minimal estimate of the relative frequency of imprecise excisions, determined in this experiment is 75%.--A successful pilot experiment is described that utilizes dysgenic perturbation of the sdry+ allele to select for small deletions of the 5' noncoding region of the rosy locus.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.     

Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.     

Identification of the in vivo and in vitro phosphorylation sites of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase appears to be unique in that it extends 24-26 residues beyond the COOH-terminal amino acid of other mammalian fructose 1,6-bisphosphatases and this extension contains phosphorylation sites. Using as a frame of reference the 335-residue sequence of pig kidney fructose 1,6-bisphosphatase (Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165), the rat liver enzyme would extend to residue 361. Limited proteolysis in the COOH-terminal region of the molecule with chymotrypsin, trypsin, or both sequentially, led us to establish that the phosphorylation sites are located at Ser residues 341 and 356. The in vitro phosphorylation of purified rat liver fructose 1,6-bisphosphatase by the catalytic subunit of cyclic AMP-dependent protein kinase results in modification at both residues, although the major site of phosphorylation (61%) is at Ser-341. In contrast, rat liver fructose 1,6-bisphosphatase purified from animals that had been injected with [32P] phosphate contains most of the label (81%) at Ser-356.     

Prostaglandin production by dispersed granulosa and theca interna cells from porcine preovulatory follicles

Prepubertal gilts were treated with 750 IU pregnant mares' serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa (GC) and theca interna (TIC) cells were prepared by microdissection and enzymatic digestion from follicles obtained 36, 72 and 108 h after PMSG treatment and incubated for up to 6 h in a chemically defined medium in the presence or absence of arachidonic acid, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and indomethacin. Production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. Both GC and TIC had the capacity to produce prostaglandins, with production by each cell type increasing markedly with follicular maturation. PGE was the major prostaglandin produced by both cellular compartments. Only PGE production by GC was consistently enhanced by addition of arachidonic acid to the incubation medium. Neither cell type was responsive to FSH and LH in vitro. Indomethacin inhibited the production of PGE and PGF by both cell types. These results provide convincing evidence for an intrafollicular source of prostaglandins and indicate that both cellular compartments contribute significantly to the increased production of prostaglandins associated with follicular rupture.