A three-step model for the rearrangement of the chloroplast trnK-psbA region of the gymnosperm Pinus contorta.

A region of the Pinus contorta chloroplast genome which contains a duplication of the psbA gene was characterized. From previous experiments it was known that the two copies of the psbA gene were located approximately 3.3 kilobase pairs (kbp) apart, that they had the same orientation and that one endpoint of the duplication was 19 base pairs (bp) downstream of the psbA stop codon. In order to determine the size and additional genetic content of the duplicated segment, both copies as well as the intervening DNA were sequenced completely. It was found that the duplicated segment was 1969 bp long, that the two copies were completely identical and were separated by 2431 bp. The duplicated segment carried, in addition to psbA, the 3' exon of the trnK gene, which was partially included in a 124 bp direct repeat. The translocated copy of the duplicated segment was found to be inserted upstream of the trnK(UUU) gene and was immediately followed by a repeated 41 bp stretch from the psbA coding region. The trnK gene was split by a 2509 bp intron which contained an open reading frame of 515 codons. Sequence comparisons of the duplicated segment and its flanking DNA to the corresponding regions of P. sylvestris, a species which lacks the rearrangements found in P. contorta, made it possible to identify 3-9 bp homologies within which recombinations had occurred. A model was derived which would accommodate the conversion of a trnK-psbA locus of the ancestral P. sylvestris-like organization into the rearranged structure found in P. contorta.     

Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.     

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.     

Neuropeptides in free-living and parasitic flatworms (Platyhelminthes). An immunocytochemical study

The nervous systems of the turbellarians Microstomum lineare and Polycelis nigra and of the cestodes Diphyllobothrium dendriticum and Schistocephalus solidus were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the neuropeptides FMRF-amide, vasotocin, leu-enkephalin, met-enkephalin, neurotensin, somatostatin, and VIP, and to the bioamine serotonin. Anti-FMRF-amide positive perikarya and fibers occurred in all species, while the occurrence of the vertebrate brain-gut peptides and serotonin varied between the species. Anti-somatostatin and anti-VIP gave a negative result. Anti-FMRF-amide and anti-vasotocin positive immunoreactivity was found in the brain and gut of M. lineare, and in the CNS and the peripheral nerve net of the cestodes. We suggest that the brain-gut peptides of free-living flatworms act on the subtegumental region in the cestodes, which lack a gut but absorb their nutrients directly through the tegument.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.     

Acclimation Processes in the Light-Harvesting System of the Cyanobacterium Anacystis nidulans following a Light Shift from White to Red Light

Cyanobacteria acclimate to changes in light by adjusting the amounts of different cellular compounds, for example the light-harvesting macromolecular complex. Described are the acclimatization responses in the light-harvesting system of the cyanobacterium Anacystis nidulans following a shift from high intensity, white light to low intensity, red light.

The phycocyanin and chlorophyll content and the relative amount of the two linker peptides (33 and 30 kilodaltons) in the phycobilisome were studied. Both the phycocyanin and chlorophyll content per cell increased after the shift, although the phycocyanin increased relatively more. The increase in phycocyanin was biphasic in nature, a fast initial phase and a slower second phase, while the chlorophyll increase was completed in one phase. The phycocyanin and chlorophyll responses to red light were immediate and were completed within 30 and 80 hours for chlorophyll and phycocyanin, respectively. An immediate response was also seen for the two phycobilisome linker peptides. The amount of both of them increased after the shift, although the 33 kilodalton linker peptide increased faster than the 30 kilodalton linker peptide. The increase of the content of the two linker peptides stopped when the phycocyanin increase shifted from the first to the second phase. We believe that the first phase of phycocyanin increase was due mainly to an increase in the phycobilisome size while the second phase was caused only by an increase in the amount of phycobilisomes. The termination of chlorophyll accumulation, which indicates that no further reaction center chlorophyll antennae were formed, occurred parallel to the onset of the second phase of phycocyanin accumulation.

     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.