In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

Computer-assisted grading of adenocarcinoma in prostatic aspirates

Conventional cytologic grading of fine needle aspirates of prostatic adenocarcinoma has been shown neither to be reproducible nor to correlate well with histologic grading. This study developed a tumor grade classification based on computerized cytomorphometric features and compared the results to conventional grading of companion tissue sections. The image analysis system evaluated architectural features of the aspirates (mainly cell cluster features and interrelationships) as well as nuclear features. Thirty-five prostatic adenocarcinomas (8 well, 19 moderately and 8 poorly differentiated) were evaluated. Discriminant functions based on data collected at medium and high resolution distinguished between aspirates from low-grade (well-differentiated) and high-grade (poorly differentiated) adenocarcinomas with 81% accuracy. Moderately differentiated cancers could not be classified as a distinct group. This study suggests that accurate grading of prostatic adenocarcinoma in fine needle aspirate smears requires the evaluation of medium-resolution features related to specimen cellularity and uniformity or crowding of cell clusters as well as of high-resolution features of nuclear area, perimeter and coarseness of chromatin texture. These findings are compared to those of other schemes for the cytologic grading of prostatic aspirates.     

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.     

Mechanosensory interneurons (MSIs) in the crayfish 6th abdominal ganglion are inhibited by activation of other MSIs

1. Identified mechanosensory interneurons (MSIs) in the 6th abdominal ganglion of the crayfish Procambarus clarkii have been shown to inhibit other projecting MSIs. 2. Interneurons sensitive to water-current stimulation of the tailfan, and which inhibited the tactile response of other MSIs when activated by depolarizing currents, were identified by iontophoresis of fluorescent dye. 3. Ten inhibitory interneurons have been identified, including both non-adapting, directional cells and phasic "touch" cells. 4. Inhibition triggered by activation of the identified cells was not widespread among fibers in the connectives. 5. Inhibition recorded intracellularly was mediated by compound inhibitory postsynaptic potentials of long duration (300-400 msec) and latencies of 13-15 msec, and therefore was apparently polysynaptic. 6. Depolarization and/or activity in MSIs, which modulates the stimulus response characteristics of related cells is a possible mechanism for contrast enhancement among directional or frequency-selective interneurons.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction.     

Effects of CaSO4 and NaCl on growth and nitrogen fixation ofLeucaena leucocephala

Effects and interactions of varying CaSO₄ and NaCl levels on growth and nitrogen fixation ofLeucaena leucocephala K8 were examined. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM) in randomized blocks with five replications. While NaCl significantly reduced plant growth, additions of CaSO₄ increased plant height, leaf number, and biomass of salt treated plants. For the nonsaline treatments, high CaSO₄ levels slightly depressed growth, which contradicts suggestions that Leucaena has a high calcium requirement. A significant calcium/sodium interaction was not seen for nodule number or weight. Nodule number was significantly depressed by 100 mM NaCl and nodule weight of the salt stressed plants significantly increased as CaSO₄ concentration increased from 0.5 to 2.5 mM. Effects of NaCl and CaSO₄ on nitrogen content of plant parts were inconclusive. The promotion of Leucaena salinity tolerance by addition of CaSO₄ may be attributed to the effect of calcium in maintaing the selective permeability of membranes.