Forskolin stimulation of pepsinogen secretion from frog esophageal mucosa is partly mediated by intrinsic cholinergic neurons

Forskolin was found to stimulate pepsinogen secretion from frog esophageal mucosa. The stimulation was dose-dependent and was accompanied with a great increase in tissue cAMP content. The response to forskolin mimicked the action of bethanechol and was not additive with bethanechol. The stimulatory effect of forskolin was inhibited by 50% in the presence of either atropine or tetrodotoxin. On the other hand, incubation in a calcium-free medium not only reduced the response to forskolin by 45% but also eliminated the influence of atropine and tetrodotoxin. These results indicate that forskolin may stimulate pepsinogen secretion from the frog esophageal mucosa via activating adenylate cyclase, and part of its effect may arise from eliciting acetylcholine release from the intrinsic neurons.     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Citric acid production by Aspergillus niger immobilized on polyurethane foam

Summary Citric acid was produced using Aspergillus niger immobilized on polyurethane foam in a bubble column reactor. Most of the adsorbed cells remained on the support and, as a result, high oxygen tension was maintained during the reactor operation. However, uncontrolled growth of the pellets made continuous reactor operation difficult. The citric acid productivity obtained from 15 vol.% foam particles containing immobilized cells was 0.135 g/l per hour. This productivity of immobilized cells was almost the same as that of free cells. The oxygen level dropped to half saturation in 5 days in the immobilized cell culture in contrast to 2 days in the free cell culture.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

DNA loop domains in mammalian spermatozoa

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46±7 kb in sperm as compared with 16±11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.     

Characterization of Developmentally Regulated cAMP/Ca2+-Independent Protein Kinases from Dictyostelium discoideum

Cell-free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate-associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S-300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE-Sephacel and heparin-Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE-Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.