Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.     

Comparison of properties of commercially available crystalline native and recombinant firefly luciferases

Commercially available crystalline native and recombinant firefly luciferases were compared. The two types of luciferase had indistinguishable responses to variation in ATP and luciferin concentrations and to omission of reaction components. The time courses of light production, the responses to nucleotide analogues, and the stability of the enzymes under several storage conditions were identical. The native enzyme had a slightly greater specific activity and was more sensitive to trypsin degradation. These differeces are probably attributable to differences in conformation.     

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

An analog of ACTH/MSH (4–9), ORG-2766, reduces cerebral uptake of morphine

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.     

Immunogenic fractions of Cryptococcus neoformans

Cryptococcus neoformans cell and culture supernatant extracts were fractionated by ion exchange and gel filtration column chromatography. Various fractions were used to immunize mice, and to assess the release of migration inhibition factor, delayed type hypersensitivity, and protective immunity after challenge with C. neoformans. Results suggest that the C. neoformans fractions, which protect mice, contain a high molecular weight, predominantly carbohydrate antigen that can be distinguished from the capsular polysaccharide.     

Purification and Characterization of an Autolysin from Clostridium acetobutylicum

A proteinaceous substance with antibiotic-like activity, resembling that of a bacteriocin, was isolated from an industrial-scale acetone-butanol fermentation of Clostridium acetobutylicum. The substance, purified by acetone precipitation, diethylaminoethyl cellulose chromatography, and polyacrylamide gel electrophoresis, was characterized as a glycoprotein with a molecular weight of 28,000. The glycoprotein was partially inactivated by certain protease enzymes. It had no effect on deoxyribonucleic acid, ribonucleic acid, or protein synthesis, and it did not result in the loss of intracellular adenosine triphosphate. The glycoprotein lysed sodium dodecyl sulfate-treated cells and cell wall preparations, and therefore it is referred to as an autolysin. The autolysin gene appeared to be chromosomal since plasmid deoxyribonucleic acid was not detected in the C. acetobutylicum strain.     

Selective-Differential Medium for Isolation and Differentiation of Pectinatus from Other Brewery Microorganisms

An agar medium, LL-agar (lactate-lead acetate) was designed to selectively differentiate members of the genus Pectinatus (S. Y. Lee, M. S. Mabee, and N. O. Jangaard, Int. J. Syst. Bacteriol. 28:582-594, 1978; S. Y. Lee, M. S. Mabee, N. O. Jangaard, and E. K. Horiuchi, J. Inst. Brew. 86:28-30, 1980) from other brewery microorganisms. Selectivity was achieved by the use of sodium lactate as the sole source of carbon and phenylethyl alcohol as an inhibitor for aerobic gram-negative bacteria and yeast. Differentiation was established by the introduction of lead acetate into the medium, which reacted with the H₂S liberated by Pectinatus and resulted in a blackening of the Pectinatus colonies while the other brewery organisms, when present, remained white. In combination with the Lee tube (J. E. Ogg, S. Y. Lee, and B. J. Ogg, Can. J. Microbiol. 25:987-990, 1979) and this medium, isolation of Pectinatus organisms from beer samples was accomplished with convenience and simplicity.     

Detached leaf enrichment: a method for detecting small numbers of Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria in seed and symptomless leaves of tomato and pepper

A method for detecting 10¹-10² cells of phytopathogenic bacteria ( Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. vesicatoria ) in either tomato or pepper seed was developed. The method is based on the enrichment of the compatible pathogen inside a detached leaf of its host when placed on a water agar medium. It was found to be superior to the diagnostic growth media method commonly used and to permit the detection of the pathogens in symptomless plants.