Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

A method for quantitation of protein in the presence of Percoll

An electromagnet was modified for measurements of the magnetic circular dichroism of samples held at cryogenic temperatures using a standard laboratory cryostat. The external dimensions of the cryostat are too great to permit its insertion in the air gap between the poles of the magnet without an unacceptable reduction in the strength of the magnetic field at the sample. This problem was overcome by designing new pole caps which become an integral part of the vacuum system of the cryostat. The ends of the new pole caps project into the body of the cryostat so that the gap between them is 1 in. or less, thus achieving a magnetic field exceeding one Tesla at the sample. No permanent alterations of the cryostat are required. The chief advantages of this design are economy and flexibility since a general purpose cryostat is used instead of a special unit designed to fit in the small space between the poles of an unmodified magnet. The cryostat used in this design cools the sample by conduction; thus the problem of optical distortions resulting from bubbling of liquid nitrogen or other cryogen is avoided and the temperature can be varied continuously using standard auxiliary equipment. Extra windows at 90° with respect to the optical beam permit inspection of the sample in situ and could be used for experiments such as fluorescence-detected magnetic circular dichroism which require optical access perpendicular to the direction of the magnetic field.     

Fermentative Conversion of Cellulose to Acetic Acid and Cellulolytic Enzyme Production by a Bacterial Mixed Culture Obtained from Sewage Sludge

A simple procedure that uses a cellulose-enriched culture started from sewage sludge was developed for producing cellulolytic enzymes and converting cellulose to acetic acid rather than CH₄ and CO₂. In this procedure, the culture which converts cellulose to CH₄ and CO₂ was mixed with a synthetic medium and cellulose and heated to 80°C for 15 min before incubation. The end products formed were acetic acid, propionic acid, CO₂, and traces of ethanol and H₂. Supernatants from 6- to 10-day-old cultures contained 16 to 36 mM acetic acid. Cellulolytic enzymes in the supernatant were stable at 2°C under aerobic conditions for up to 4 weeks and had the ability to hydrolyze carboxymethyl cellulose, a microcystalline cellulose, cellobiose, xylan, and filter paper to reducing sugars.     

1-Hydroxycanthin-6-one,an alkaloid from Ailanthus giraldii

A new alkaloid has been isolated from the heartwood of Ailanthus giraldii and its structure determined as 1-hydroxycanthin-6-one.     

Isolation and structure of 13,18-dehydroexcelsin,a quassinoid,and glaucarubol from Ailanthus excelsa

A new quassinoid, 13,18-dehydroexcelsin and glaucarubol have been isolated from the bark of Ailanthus excelsa.     

Treatment of refractory congestive heart failure and normokalemic hypochloremic alkalosis with acetazolamide and spironolactone.

Combination therapy with a loop diuretic and an aldosterone antagonist can produce normokalemic hypochloremic alkalosis, a complication not previously documented in the literature. This report describes 74 patients who had severe congestive heart failure treated with a combination of furosemide and spironolactone in whom this complication developed. Acetazolamide corrected the metabolic abnormality. The combination of furosemide and spironolactone with intermittent courses of acetazolamide was very effective in the treatment of severe congestive heart failure complicated by normokalemic hypochloremic alkalosis.     

Transport of methylamine by Pseudomonas sp. MA.

Pseudomonas sp. MA grows on methylamines as a sole source of carbon, nitrogen, and energy. The transport of methylamine into the organism was investigated. It was found that this organism possesses an inducible transport system for methylamine having the following physical parameters: pH optimum, 7.2; temperature optimum, 30 to 35 degrees C; Km, 1 to 30 mM; Vmax, 90 to 120 nmol/min per mg (dry weight) of cells. Methylamine uptake was curtailed by azide, cyanide, and carbonyl cyanide-m-chlorophenylhydrazone; osmotic shock treatment reduced the uptake by 50%. The uptake was not effectively inhibited by ammonium ion, amino acids, or amides, but was competitively inhibited by short-chain alkylamines. Cells grown on succinate-ammonium chloride did not possess the transport system, but it could be induced in such cells by methylamine in 20 h. Cells grown with methylamine as a sole nitrogen, but not carbon, source transported methylamine at a reduced rate.