Identification,cloning, and sequencing of DNA essential for encapsulation of Streptococcus pneumoniae

This paper reports the cloning and sequencing of a region of DNA from Streptococcus pneumoniae serotype 3 surrounding transposon Tn916, insertion of which was previously shown to result in lack of expression of the extracellular capsule. Sequence analysis revealed that the transposon inserted into a consensus insertion site 71 bp from the 5 end of the cloned fragment. Within the clone, 3 downstream regions from two different pneumococcal lytA genes were identified, as well as a putative 194 AA open reading frame (ORF1). Moreover, two copies of the repeat element BOX, oriented in opposite directions, were located immediately 3 of orf1. Within the region bounded by the first pair of internal sequencing primers, analysis revealed that the fragment amplified by PCR was always of the same size. Moreover, Southern blotting showed that for all serotypes examined to date, homology exists with the cloned fragment. These results indicate that this region of the chromosome is highly conserved and, taken together with other independently derived data, suggest that interruptions or deletions within this DNA lead to unencapsulation.     

Cell surface topography of Candida and Leucosporidium yeasts as revealed by scanning electron microscopy.

The cell surface topography of the following yeast strains was examined by scanning electron microscopy: Candida slooffii, C. lipolytica, Leucosporidium frigidum, and L. nivalis. Multipolar and lateral budding were observed in the Candida yeasts in contrast to bipolar budding in the Leucosporidium species. The cell surface topography and the morphology of the bud and birth scars in these yeasts differed markedly. Apart from the bud and birth scars, the cells of C. slooffii showed a relatively smooth topography. The bud scars were seen as a circular ridge of wall material surrounding a markedly convex scar plug. Birth scars were raised, rounded structures, which appeared to distend upon cell growth. In contrast, bud scars of C. lipolytica were platelike, lacked a distinct annulus of wall material, and were much less protuberent than those of C. slooffii. Birth scars were a more permanent feature of these cells. The topography of Leucosporidium yeasts was characterized by the presence of numerous protrusions on the cell surface. In some cases, the entire cell surface was covered by these protrusions. There appeared to be some correlations between the age of the cell and the extent of surface protrusions and degree of surface convolution...     

Does cyclic GMP mediate amylase release from mouse parotid acini?

In mouse parotid acini both cholinergic and beta-adrenergic agonists increased intracellular levels of cyclic-GMP (c-GMP) as well as amylase release. The derivative of c-GMP, 8-bromo-c-GMP, mimicked the effects of cholinergic and beta-adrenergic stimulation on amylase release. Nitroprusside (NP), hydroxylamine (HA) and sodium azide (NaA) increased c-GMP levels and also enhanced amylase release in a dose-dependent manner; cyclic-AMP (c-AMP) levels were not affected. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (MIX) enhanced the effects of carbachol on both c-GMP accumulation and amylase release. These results suggest that c-GMP may mediate the actions of cholinergic agonists and at least partially mediate the actions of beta-adrenergic agonists on mouse parotid enzyme release.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

Effects of N-serve (2-chloro-6-(trichloromethyl)pyridine) formulations on nitrification and on loss of nitrate in sand culture experiments

Summary N-serve (2-chloro-6-(trichloromethyl)pyridine) was tested as an inhibitor of nitrification of ammonium or urea in sand cultures. Nitrification was reduced but not prevented by N-Serve present at between 5 and 20 ppm in solution or by weight of sand. In the presence of root debris and acetone, used in some experiments at 2–4 ml/l of nutrient to convey N-Serve, denitrification was stimulated under the same conditions and resulted in loss of a large proportion of nitrate, probably mainly as gaseous products and some nitrite. These losses were greater when N-serve was also present. There was also conversion of nitrate to an insoluble form in the sand. A smaller proportional loss of nitrate occurred in other treatments in the presence of root debris when N-Serve was added without acetone, either as the commercial formulation 24E or as a solid. Thus, using N-Serve to inhibit nitrification may encourage denitrifying organisms especially in the presence of carbon sources including root debris or acetone. Large decreases of nitrate reductase activity in plants produced by using N-Serve in the presence of ammonium or urea were caused as much by losses of nitrate in the presence of acetone as by prevention of nitrate formation. Other N-Serve treatments (solid or 24E) decreased enzyme induction by between 50 and 90 per cent as a result mainly of reduced nitrification.     

Origins of the Human Genome Project

The Human Genome Project has become a reality. Building on a debate that dates back to 1985, several genome projects are now in full stride around the world, and more are likely to form in the next several years. Italy began its genome program in 1987, and the United Kingdom and U.S.S.R. in 1988. The European communities mounted several genome projects on yeast, bacteria, Drosophila, and Arabidospis thaliana (a rapidly growing plant with a small genome) in 1988, and in 1990 commenced a new 2-year program on the human genome. In the United States, we have completed the first year of operation of the National Center for Human Genome Research at the National Institutes of Health (NIH), now the largest single funding source for genome research in the world. There have been dedicated budgets focused on genome-scale research at NIH, the U.S. Department of Energy, and the Howard Hughes Medical Institute for several years, and results are beginning to accumulate. There were three annual meetings on genome mapping and sequencing at Cold Spring Harbor, New York, in the spring of 1988, 1989, and 1990; the talks have shifted from a discussion about how to approach problems to presenting results from experiments already performed. We have finally begun to work rather than merely talk. The purpose of genome projects is to assemble data on the structure of DNA in human chromosomes and those of other organisms. A second goal is to develop new technologies to perform mapping and sequencing. There have been impressive technical advances in the past 5 years since the debate about the human genome project began. We are on the verge of beginning pilot projects to test several approaches to sequencing long stretches of DNA, using both automation and manual methods. Ordered sets of yeast artificial chromosome and cosmid clones have been assembled to span more than 2 million base pairs of several human chromosomes, and a region of 10 million base pairs has been assembled for Caenorhabditis elegans by a collaboration between Washington University and the Medical Research Council laboratory in Cambridge, U.K. This project is now turning to sequencing C. elegans DNA as a logical extension of this work. These are but the first fruits of the genome project. There is much more to come.     

Detection of microgram quantities of carrier ampholytes in electrofocused proteins by thin-layer chromatography

A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an R_f greater than 0.50. We found that 1.25 μg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M_r cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenic exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).