Mutants of Saccharomyces cerevisiae That Incorporate Deoxythymidine-5′-Monophosphate Into Deoxyribonucleic Acid In Vivo

Spontaneous mutants of Saccharomyces cerevisiae able to incorporate deoxythymidine-5′-monophosphate (dTMP) into deoxyribonucleic acid (DNA) have been selected based on their ability to grow in the presence of aminopterin and sulfanilamide if dTMP is present. Essentially all mutants (called tup) selected in this way required dTMP for growth in the presence of the two drugs, but none required dTMP in the absence of the drugs. Neither thymine nor thymidine would satisfy this requirement. Equimolar amounts of ³²P- and ³H-base-labeled dTMP were incorporated by the mutants into alkali-stable, deoxyribonuclease-sensitive material. In the presence of aminopterin and sulfanilamide, this incorporation was sufficient to account for a substantial proportion of the thymine residues in the cellular DNA, whereas in the absence of the drugs only about 40% as much of the thymine residues originated from the medium. Of 29 mutants examined, all were recessive and 17 showed 2:2 segregation in crosses with a wild-type strain. The lesions in these mutants fell into four complementation groups: one (tup1) occurs on chromosome III; another (tup3) is on chromosome II; and a third (tup4) was centromere linked. Strains of the genotype α tup1 mated with lower than normal efficiency with a strains, but with higher than normal efficiency with α strains. Strains of genotype a/α tup1/tup1 failed to sporulate, whereas homozygous diploids for tup2, tup3, or tup4 sporulated normally, as did a/α tup1/+ strains.     

The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of (45)Ca(2+) transport by rat liver mitochondria. This method was used in conjunction with studies of Ca(2+)-stimulated respiration to examine the mechanisms of inhibition of Ca(2+) transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca(2+) transport non-competitively with K(i) 3x10(-8)m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La(3+) is competitive (K(i)=2x10(-8)m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ;low-affinity' Ca(2+)-binding sites). La(3+) competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La(3+). The results are discussed briefly in relation to possible models for a Ca(2+) carrier.     

Effect of Lytic Enzymes of Acanthamoeba castellanii on Bacterial Cell Walls

Extracts of Acanthamoeba castellanii (Neff) contain alpha- and beta-glucosidase, beta-galactosidase, beta-N-acetylglucosaminidase, amylase, and peptidase. All of these activities are optimal between pH 3 and 4. These extracts also were found to clarify suspensions of cell walls from nine different gram-positive bacteria, including Micrococcus lysodeikticus. The pH optimum for the lytic activity was between 3 and 4. The extent of lysis of the various cell walls did not correlate with the release of free amino groups and of free N-acetylated sugars from the walls during digestion with these extracts. Suspensions of cell walls of Escherichia coli (a gram-negative bacterium), Cordiceps militaris (a fungus), and Acanthamoeba cysts, as well as of colloidal chitin, were not clarified by incubation with these extracts, although reducing sugars were released from each of these materials. Exhaustive digestion of M. lysodeikticus walls by lysozyme released no free N-acetylglucosamine. The products of exhaustive digestion of this cell wall with Acanthamoeba extracts were free N-acetylglucosamine, free N-acetylmuramic acid, glycine, alanine, glutamic acid, lysine, and N-acetylmuramic acid peptide fragments. These results suggest that the amoeba extracts contain endo- and exo-hexosaminidases, in addition to beta-hexosaminidase and peptide hydrolases.     

NON-INFECTIOUS INFLAMMATORY AND NEOPLASTIC DISORDERS OF THE MALE GENITALIA

The cutaneous manifestations of the male external genitalia are difficult to diagnose. They may be associated with systemic disease (Reiter''s disease, psoriatic arthritis, angiokeratoma corporis diffusum). In dealing with a lesion of this area that does not heal, adequate biopsy is advisable to rule out malignant disease (Bowen''s disease, melanoma, Kaposi''s sarcoma, Paget''s disease, erythroplasia).     

Ketamine-induced tongue protrusions in rats

1. The purpose of this study was to demonstrate that ketamine anesthesia (100 mg/kg) induces tongue protrusions (P) in addition to retrusions (R) and swallows (S) in adult rats. 2. These linguo-pharyngeal events occur alone or combined in various sequential patterns. 3. The SPR sequence is not the predominant pattern in all preparations suggesting profound disruption of physiological linkages by ketamine. 4. Haloperidol administration suppresses these events for 1-120 min depending on the dose (0.75-2.5 mg/kg). 5. Swallows are the least vulnerable to haloperidol. 6. This and previous findings provide further evidence that ketamine induced linguo-pharyngeal activity can serve as a model for acute or tardive dyskinesia better than stereotypies.     

Effects of medium composition on penicillin-induced hydrolysis and loss of RNA and culture turbidity in group A streptococci.

Exposure to penicillin G of exponentially growing cultures of group A streptococci growing in chemically defined medium (CDM) can lead to extensive loss of culture turbidity. Significant reductions in culture turbidity did not accompany comparable treatments of group A streptococci growing in Todd-Hewitt broth (THB). Studies with THB and a high-molecular-weight (greater than 12,000) fraction of THB demonstrated that components in this complex medium inhibited the efflux of RNA hydrolysis products from otherwise intact cells. Hydrolysis products accumulated intracellularly and inhibited the extensive hydrolysis of RNA and consequently the loss of culture turbidity. Results of survival studies with cultures of group A streptococci exposed to penicillin G in THB demonstrated that this treatment protocol produces conditions of phenotypic tolerance relative to exposure in CDM. In combination, these findings provide further support for the hypothesis of RNA hydrolysis as the bactericidal mechanism of penicillin G action in this nonlytic death phenotype.