The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them.

The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium. We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol. wt 99,000), beta- (mol. wt 30,000) and gamma- (mol. wt 27,500) subunits. The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated. The N-terminal amino acid sequence of each subunit has been determined. Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits. The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.     

A comparison of anionic sites in the glomerular basement membranes from different classes of fishes

Summary Cationized ferritin was injected into the circulatory system of teleosts, the sea raven and Atlantic eelpout, and into elasmobranchs, the spiny dogfish and the skate, to determine if the glomerular basement membranes (GBM) from these different groups of fishes possess anionic binding sites similar to those present in the GBM of mammals. The distribution of cationized ferritin was the same in all fishes listed. Cationized ferritin was localized only in the GBM and the mesangial matrix. The regular distribution of cationized ferritin within the laminae rarae (60 nm intervals) was taken as evidence of the presence of anionic binding sites. Cationized ferritin did not bind to the glomerular capillary endothelium, nor was any of it localized at the base of the slit diaphragms of the foot processes of the podocytes. The distribution of binding sites in the GBM of these fishes is similar to that in another teleost, the winter flounder, and in a cyclostome, the hagfish.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

Summary A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles.Within the central ganglia many neurones, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata.Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Anaerobic biodegradation of phenolic compounds in digested sludge.

We examined the anaerobic degradation of phenol and the ortho, meta, and para isomers of chlorophenol, methoxyphenol, methylphenol (cresol), and nitrophenol in anaerobic sewage sludge diluted to 10% in a mineral salts medium. Of the 12 monosubstituted phenols studied, only p-chlorophenol and o-cresol were not significantly degraded during an 8-week incubation period. The phenol compounds degraded and the time required for complete substrate disappearance (in weeks) were: phenol (2), o-chlorophenol (3), m-chlorophenol (7), o-methoxyphenol (2), m- and p-methoxyphenol (1), m-cresol (7), p-cresol (3), and o-, m-, and p-nitrophenol (1). Complete mineralization of phenol, o-chlorophenol, m-cresol, p-cresol, o-nitrophenol, p-nitrophenol, and o-, m-, and p-methoxyphenol was observed. In general, the presence of Cl and NO2 groups on phenols inhibited methane production. Elimination or transformation of these substituents was accompanied by increased methane production, o-Chlorophenol was metabolized to phenol, which indicated that dechlorination was the initial degradation step. The methoxyphenols were transformed to the corresponding dihydroxybenzene compounds, which were subsequently mineralized.     

Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing

Human lymphocytes stored in the frozen state may be thawed, placed on cytotoxicity plates, refrozen, rethawed and used for screening sera or tissue-typing of the cells. The simple procedure described uses only a ?90 °C refrigerator for both freezing and storage of the cells. The technique permits a laboratory to collect a variety of cells over a long period, so that a set of test plates with cells from 10 to 20 donors can be prepared when a convenient number of donor cells are available. Also, the refrozen cells in cytotoxicity test plates may be warmed to the temperature of dry ice for 24 hr, returned to the refrigerator set at a slightly lower temperature, and at a later time, these cells may be thawed and used for serum screening. In view of these results, it appears possible to ship the refrozen cells from one laboratory to another using simple dry ice storage during the transfer. Negative reactions due to soluble antigens in the suspending sera can be obviated by washing out these sera and replacing them with medium 199 or alternatively, fetal calf serum can be used to replace the human serum in the suspending media.     

Electron microscopy of phages for Agrobacterium tumefaciens

Summary Bacteriophages for three strains of A. tumefaciens were concentrated by ultracentrifugation, stained with 1% phosphotungstic acid (PTA), or 0.5% uranyl acetate, and examined with the electron microscope. Phage PT11 was a bacillary-shaped particle with a whip-like tail containing a knob at its distal end. Phage PIIBNV6 appeared to have an icosahedral head. The wide non-contractile tail terminated in a plate with pegs. Phage PIIBNV6-C was an icosahedral particle with a short, spike-like tail. Host cells of A. tumefaciens were encapsulated rods bearing polar or lateral flagella.Published with approval of the Director, Wisconsin Agricultural Experiment Station, Madison, Wisconsin, U.S.A. 53706.     

Observations on the vacuolar structure of the human syncytiotrophoblast

Summary The literature on the vacuolar structure of the human syncytiotrophoblast is briefly reviewed. Personal observations based on light and electron microscopic investigations are described and illustrated. The nature of the different kinds of syncytial vacuolation is discussed. In particular dilated vacuoles, which may be very large indeed, receive special attention; situated adjacent to the syncytial nuclei they have been called juxtanuclear vacuoles. They are directly continuous with the perinuclear spaces, and often receive tubular communications from the endoplasmic reticular system. The possible functional role of the syncytial vacuolar system is discussed in the light of the authors' findings and the related literature.Dedicated to Professor Dr. Kurt Goerttler on the occasion of his seventieth birthday.