Induction and characterization of the major surfactant apoprotein during rabbit fetal lung development

Antibodies directed against the major apoprotein associated with rabbit lung surfactant were used to characterize the induction and cellular localization of this protein during rabbit fetal lung development. In lung tissues from rabbits of 26 days gestational age and older, discrete epithelial type II cells were stained positively using the peroxidase antiperoxidase technique. The content of the major protein in homogenates of fetal lung tissue was analyzed using an immunoblotting technique. A protein of about 29 kDa, pI less than or equal to 5.6, was first detectable in fetal lung tissue on day 24 of gestation. The 29-36 kDa, mature form of the surfactant apoprotein was first detectable in lung homogenates from 30-day gestational age fetal rabbits. Treatment of homogenates of day 26 and 31 fetal lung tissues with endoglycosidase F, yielded, in both cases, an immunoreactive triplet with more neutral isoelectric points than the proteins in the untreated homogenates. By immunoblot analysis, we found that only the 29-36 kDa, mature form of the surfactant apoprotein was present in lamellar bodies purified from lung tissues of fetuses of 28 and 31 days and from day 2 neonates. These findings are suggestive that only the mature, 29-36 kDa form of the surfactant apoprotein is associated with lamellar bodies during fetal lung type II cell differentiation in vivo.     

Regulation by follicle-stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells

The effects of FSH to increase the activity of aromatase, as well as the synthesis of the components of the aromatase enzyme complex, have been studied in human ovarian granulosa cells obtained from women undergoing oocyte retrieval. FSH increased aromatase activity, as well as the synthesis of aromatase cytochrome P-450 (P-450AROM) in a time-dependent fashion, whereas in the absence of FSH, both activity and synthesis declined with duration of culture. The effect of FSH was mimicked by forskolin, an activator of adenylate cyclase. FSH also increased the synthesis of NADPH-cytochrome P-450 reductase, but to a relatively modest extent. The levels of hybridizable mRNA species encoding cytochrome P-450AROM of lengths 3.0, 2.4, and 1.6 kilobases were also increased with FSH treatment. It is concluded that the regulation of aromatase activity by FSH in human granulosa cells is mediated primarily by changes in the synthesis of cytochrome P-450AROM, that this action of FSH is mediated by cAMP, and that the changes in cytochrome P-450AROM synthesis are the consequences of changes in the levels of mRNA encoding this enzyme.     

Ar+ plasma-induced damage to DNA in bacteriophage lambda: implications for the arrangement of DNA in the phage head.

Bacteriophage lambda was bombarded with low-energy Ar+ ions with the goal of determining whether particular regions of the DNA genome are found preferentially in the outer portion of the packaged DNA mass. The strategy was to fragment the DNA selectively near the surface of the virus by exposing intact phage to Ar+ ions energetic enough to break covalent chemical bonds in DNA but not energetic enough to penetrate deeply beneath the viral capsid shell. Broken DNA was then isolated, and its genomic origin was identified by Southern hybridization to mapped restriction fragments of lambda DNA. Analysis of such Southern blots revealed that all regions of the lambda genome were represented among the small DNA fragments generated during all times of Ar+ bombardment examined. Depending on the duration of exposure, however, particular regions of the genome were found to be enriched in the small-fragment population. After short periods of exposure, sequences from the leftmost 10% and from the right half of the standard genetic map were enriched in the broken-DNA fraction. Among sequences in the right half of the genome, the enrichment was progressively more pronounced beginning in the middle of the genetic map and proceeding toward the right end. In phage bombarded for longer periods of time, rightward sequences were preferentially depleted in the small-fragment population. In contrast, when Ar+ bombardment was carried out with free lambda DNA rather than intact phage, small DNA fragments arose uniformly from all regions of the genome at all times of exposure examined. The results indicate that in the intact phage, DNA sequences from the right half and from the very leftmost regions of the genome have a tendency to lie closer to the capsid than does the remainder of the genome. Since DNA is packaged into the prohead beginning at the left end, our results suggest that packaging occurs in such a way that newly entering DNA tends to be disposed externally to that packaged at earlier times.     

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.     

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.      

Three-dimensional disorder of dipolar probes in a helical array. Application to muscle cross-bridges.

Fluorescence polarization and EPR experiments on azimuthally randomized helices bearing extrinsic (dipolar) probes yield information about the axial orientation and order of the probes. If the orientation of the probe on the structure bearing it is known and disorder is absent, the orientation of the structure may be ascertained. For cases where less probe orientation information is available and/or disorder is present, the available structural information is correspondingly reduced. Here we examine the available data on probes attached to cross-bridges in muscle fibers: four plausible cases of three-dimensional cross-bridge disorders are numerically modeled muscle in states of rigor and relaxation. In rigor, where the reported probe disorder is small (Thomas and Cooke, 1980), it was found that the cross-bridge disorder was also small. On the other hand, for the relaxed state where the probes are found to be completely disordered, the cross-bridges may have a considerable amount of order. This possibility is in concert with the results of x-ray diffraction, in which the presence of well-developed myosin-based layer lines indicates considerable order in relaxed muscle.     

Polarization from a Helix of Fluorophores and its Relation to that Obtained from Muscle

Expressions for the polarization of fluorescence from fluorophores in a helical array have been obtained. These predictions have been compared with experimental data obtained by reacting fluorescent S-1 subfragments of myosin (extrinsically labeled with N-(iodoacetylaminoethyl)-5-napthylamine-1-sulfonic acid [1,5-IAEDANS]) with glycerated rabbit psoas fibers in rigor. A comparison with data obtained by directly labeling fibers with this fluorophore is also presented.     

Caerulein secretion by dermal glands in xenopus laevis

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.     

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate and testosterone production in isolated Leydig cells.

Gonadotropin binding and stimulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) formation and testosterone synthesis were studied in collagenase-dispersed interstitial cells from the adult rat testis. Binding of 125I-human chorionic gonadotropin (hCG) by isolated Leydig cells was of high affinity (Ka = 10(10) M-1) and low capacity, equivalent to approximately 6000 sites/cell. The binding data were consistent with the presence of a single order of receptors, with no interaction between binding sites. Stimulation of testosterone synthesis by increasing concentrations of hCG was completely dissociated from changes in cyclic AMP formation, and maximum activation of steroidogenesis was induced by hCG concentrations which had no effect upon cyclic AMP production. Kinetic analysis of gonadotropin-induced responses in dispersed Leydig cells also showed a marked dissociation between steroidogenesis and cyclic nucleotide formation. Low concentrations of hCG caused maximum stimulation of testosterone production which was not accompanied by a rise in cyclic AMP formation at any time after addition of gonadotropin. Higher concentrations of hCG caused marked elevations of cyclic AMP at progressively earlier time intervals, but did not alter the 20 to 30 min lag period required for induction of testosterone synthesis. These observations indicated that occupancy of gonadotropin receptors occurs over a much wider range of hCG concentration than that required for maximum steroidogenesis.     

Clockwise and counterclockwise pinwheel colony morphologies of Bacillus subtilis are correlated with the helix hand of the strain

Helical macrofiber-producing strains of Bacillus subtilis grown on fresh complex medium semisolid surfaces formed "pinwheel"-shaped colonies. Clockwise pinwheel projections arose from colonies of strains that produce right-handed helical macrofibers in fluid cultures. Most strains able to make left-handed helical macrofibers in fluid grew as disorganized wavy colonies without directed projections. A phage-resistant left-handed mutant was found that produces very tight colonies with pinwheel projections that lie counterclockwise relative to the colony. The pinwheel colony morphology is interpreted therefore in terms of the cell surface organization and helical growth.