In vitro tolerability of human nasal mucosa: histopathological and scanning electron-microscopic evaluation of nasal forms containing Sandostatin®

Anin vitro human nasal model was developed as a tool to study the local tolerabiliity of nasal powder forms using excised nasal mucosa in a diffusion chamber. The suitability of this model was tested using Sandostatin^® (SMS) an octapeptide analog of somatostatin, as a reference drug enhanced by Avicel^® (microcrystalline cellulose) or lactose (100 mesh). The standard nasal spray vehicle was taken as a harmless control and 1% chenodeoxycholate (CDC) as a harmful control in terms of local tolerability. The extent of peptide permeation was determined by measuring SMS concentration in the receiving chamber. The labeling of SMS was detected by immunoperoxidase staining on cross sections. The local tolerability for all tested forms was assessed by histopathological examination and scanning electron microscopy. The apparent permeation coefficient allowed us to rank the absorption of the tested drug forms as Avicel > spray=lactose>1%CDC. For all formulations, SMS was detected in the epithelium. No changes of the nasal mucosa could be observed with Avicel, lactose or nasal spray vehicle in the presence or absence of SMS. 1%CDC with or without drug showed an immediate destruction of the nasal epithelium. The validation of thisin vitro model using human nasal mucosa will be further discussed as a tool for assessing the local tolerability of intranasally applied test substances.Abbreviations CDC chenodeoxycholate - SMS Sandostatin^® or octreotide     

Significance of nitrate leaching and long term N immobilization after deepening the plough layers for the N regime of arable soils in N.W. Germany

Field studies were conducted to assess the turnover and the leaching of nitrogen in arable soils of Lower Saxony (NW Germany). The mean surplus N (difference between N inputs by fertilization and N export by the yield; 146 field plots) from 1985–1988 amounted to 38 kg ha^-1 yr^-1 in fine textured (clay, loam, silt) and to 98 kg ha^-1 yr^-1 in coarse (sandy) soils. Leaching of nitrate calculated by a simple functional model for simulation of the N regime over the winter period (i.e. mineralization and leaching) was 16 kg ha^-1 in the fine and 63 kg N ha^-1 in coarse soils (mean values of the winter periods 1985–1988 from 256 plots).Before the 1960s, the depth of the A_p horizons rarely exceeded 25 cm in arable soils of the former FRG. During the last three decades, ploughing depth has increased to at least 35 cm. The mass balance calculations for total N after ploughing to 35 cm in loess soils of southern Lower Saxony (105 farm plots) yielded a mean increase in total N by about 900 kg ha^-1 in 20 years. With respect to soil organic matter equilibria, N accumulation will continue for at least another 10 years on 67% of the examined farm plots. This study suggests that long term N immobilization is one of the most important sinks for nitrogen in arable soils of Germany. For simulation of the N dynamics over the growing season and for long time periods total nitrogen dynamics need to be considered.     

The Rp3 disease resistance gene of maize: mapping and characterization of introgressed alleles

The Rp3 locus of maize conditions race-specific resistance to a fungal rust pathogen, Puccinia sorghi. Both morphological and DNA markers were employed to characterize alleles of Rp3 and to accurately position Rp3 on the maize genetic map. DNA marker polymorphisms distinctive to each Rp3 allele were identified, allowing the identification of specific Rp3 alleles in cases where rust races that differentiate particular alleles are not available. In a population of 427 progeny, Rp3 and Rg1 were found to be completely linked, while Lg3 was approximately 3 cM proximal on the long arm of chromosome 3. In this same population, 12 RFLP markers were mapped relative to Rp3; the closest markers were UMC102 (about 1cM distal to Rp1) and NPI114 (1–2 cM proximal). These and additional DNA probes were used to characterize the nature and extent of flanking DNA that was carried along when six different Rp3 alleles were backcrossed into a single background. Depending upon the allele investigated, a minimum of 2–10cM of polymorphic DNA flanking the Rp3 locus was retained through the introgression process. In addition, many of the probes that map near Rp3 were found to detect an additional fragment in the Rp3 region, indicating that portions of this chromosomal segment have been tendemly duplicated. The materials and results generated will permit marker-assisted entry of Rp3 into different maize backgrounds and lay the foundation for the eventual map-based cloning of Rp3.     

A New Experiment for the Measurement of nJ(C,P) Coupling Constants Including 3J(C4′i,Pi) and 3J(C4′i,Pi+1) in Oligonucleotides

A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4i,Pi) and the sequential 3J(C4i,Pi+1) for the C4 resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles and can be determined.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them.     

Laboratory measurements and simulations of ammonia volatilization from urea applied to calcareous Chinese loess soils

Ammonia volatilization is the major pathway for mineral nitrogen loss in the calcareous soils of the Chinese loess plateau, with maximum losses reaching 50% of the fertilizer-N applied. A volatilization-diffusion experiment was carried out in the laboratory using a forced-draft system and soil columns of 15.5 cm depth. Urea was surface applied at rates of 210 kg N ha^-1 to a soil with 10% CaCO₃ and a pH of 7.7. The amount of ammonia volatilized as well as the concentration profiles of ammoniacal-nitrogen and soil pH in the upper 50 mm of the soil columns after 4, 7 and 10 days were measured and subsequently modelled. The mechanistic model of Rachhpal-Singh and Nye, originally developed for neutral, non-calcareous soils, was modified to include the pH-buffering action of the soil carbonates. Model parameters were independently determined or taken from the literature. Measured and predicted cumulative NH₃ losses agreed very well in the first 10 days following fertilizer application. However, in contrast to the simulations, NH₃-volatilization was still proceeding in the experiment even after 13 days, with cumulative losses reaching 60% of the applied N. In addition to the high initial soil pH, the low bulk density and high volumetric air content of the soil columns used for the experiment proved decisive for the high rates of ammonia volatilization, provoking a strong increase in the amount of ammoniacal-N diffusing towards the soil surface as gaseous NH₃. The simulations showed that due to the high soil pH, the buffering action of the soil carbonates played a comparatively smaller role.     

Purification and properties of a mitochondrial NAD+ glycohydrolase

A 60- to 70-fold purification of an NAD⁺ glycohydrolase from the inner membrane of rat liver mitochondria to apparent homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide slab gel is described. The minimum molecular weight of the enzyme on polyacrylamide gels in the presence of SDS is around 62,000. The enzyme splits NAD⁺ to ADP-ribose and, presumably, nicotinamide. No phosphatase or phosphodiesterase activity is detected in the purified enzyme preparation. The enzyme shows high activity with NAD⁺ and moderate activity with NADP⁺ as substrates NAD(P)Hs are poor substrates. ATP and nicotinamide inhibit the enzyme. A possible participation of the enzyme in the mechanism of calcium release from rat liver mitochondria is discussed.     

Developmentally regulated RNA binding proteins during oogenesis in Xenopus laevis

During oogenesis, Xenopus oocytes synthesize and accumulate all types of RNA. In particular, they store poly(A+) RNA to such an extent that only about 5% is actually translated in the oocyte. Using a protein blotting and in vitro binding assay, we have identified proteins which are associated with poly(A+) RNA and perhaps other RNAs as well. Two groups of binding proteins were identified. The first group accumulates during oogenesis, generally is less than 50,000 molecular weight, and sediments in the 80 S and polysome regions of a gradient. These proteins most likely include ribosomal proteins. A second group of proteins is oocyte-specific, sediments less than 80 S as well 80 S and slightly heavier, generally has molecular weights greater than 50,000, and diminishes in amount as oogenesis progresses. In addition, these proteins are retained by oligo(dT)-cellulose when ribonucleoproteins are analyzed by chromatography and, when challenged with several different types of RNA in vitro, bind poly(A+) RNA preferentially. The possibility that some of these proteins might regulate the stability or translatability of mRNAs during oogenesis is discussed.     

Blue light control of the level of two plastid mRNAs in cultured plant cells

Summary In suspension cultured callus cells of tobacco (Nicotiana tabacum var. Samsun) the development of chloroplasts is strictly blue light-dependent. During this process chlorophylls and other pigments as well as membrane and stroma proteins are synthesized de-novo. Cloned chloroplast genes of mustard encoding the large subunit (LSU) of ribulosebisphosphate carboxylase/oxygenase (EC. 4.1.1.39; RuBPCase) and the precursor polypeptide of the 32-kD membrane protein were used to study the effect of blue light on the steady-state concentration of the complementary mRNA sequences. For both a rapid increase in dark-grown cells in response to blue light-irradiation was detected by RNA dot-hybridization technique. The time courses coincide with those previously elucidated for the synthesis rates of both LSU and the membrane protein. The results support the notion that blue light acts primarily through mRNA induction.