Robert J. Rutman

Bipiperidyl Mustard, A New Obesifying Agent in the Mouse

Bipiperidyl mustard, after conversion to its bis-cyclic immonium ion forme, has been found to cause rapid and extensive lipid deposition in mice given single doses of the drug. The obe sity appears to result from an alkylation reaction; the responses of the treated ani mals to food restriction and realimenta tion resemble those observed in gold thioglucose-treated mice; also in anal ogy to gold thioglucose, the bipiperidyl mustard effects can be counteracted by sulfhydryl compounds. These observa tions are suggestive of a similarity of mode of action between the alkylating agent and gold thioglucose, a conclusion which is supported by preliminary find ings of ventromedial lesions in the hypo thalamus of the bipiperidyl mustard treated mice.

Observations on the mechanism of the alkylation reaction between nitrogen mustard and DNA

The alkylation and cross-linking of DNA by nitrogen mustard proceeds as two pseudo-first-order reactions; at 37° no unstable displaceable intermediates are observed, but the cross-linking reaction can be interrupted by nucleophilic competition. At 0°, the rate of the cross-linking reaction is greatly reduced, and the sequential steps can be completely dissociated. Under these circumstances, labile intermediates, displaceable by nucleophilic competition, are accumulated. The nature of the low-temperature intermediate is unknown at present; our data, however, suggest the need for further examination of the proposal of Lett, Parkins and Alexander1 involving transalkylation from triester phosphate to N-alkyl linkages.

A comparison of the effects of alkylation on the DNA of sensitive and resistant Lettre-Ehrlich cells following in vivo exposure to nitrogen mustard

Nitrogen mustard-sensitive and resistant Lettre-Ehrlich cells have been compared as to the effects of in vivo recovery on the DNA. It has been observed that: 1. 1. Both sensitive and resistant cells rapidly open cross-links and remove alkylated bases from their DNA, the rates being indistinguishable for the two cell strains. 2. 2. Both cell types preferentially excise N7-bis-diguaninyl alkylation products as compared to total as well as to N7-mono-guaninyl alkylations. 3. 3. Sensitive cells show a rapid and uncoordinated nicking of their DNA after alkylation and a failure to rejoin the broken ends. In contrast, resistant cells show a coordinated process of excision and repair. Our data, taken in conjunction with those of Walker I. G. and Reid, D. B. (1971) Cancer Res. 31, 510, suggest the existence of various repair deficiencies in mammalian cells, analogous to repair specific ligase and endonuclease deficiencies in microorganisms.

EFFECT OF METAL IONS ON IN VITRO GLUCONEOGENESIS IN RAT KIDNEY CORTEX SLICES.

The effect of metal ions on glucose formation from amino acids and glycolytic and tricarboxylic acid cycle intermediates has been examined in rat kidney cortex slices in vitro. Of the metals tested, only Mn ++ and Ca ++ have been shown to be stimulatory, while Zn ++ , Cu ++ , and Cd ++ are inhibitory. The case of Mn ++ activation is of particular interest because Mg ++ ions are inactive in this system, despite the similarities usually observed in the in vitro systems. The stimulation of gluconeogenesis from α-keto acids is comparable for both Ca ++ and Mn ++ , in contrast to the lack of a Mn ++ effect with the homologous l-α-amino acids. Evidence is presented as to the possible significance of metal ions in regulating carbohydrate metabolism.

Poly adenylic acid sequences in mitochondrial RNA

Abstract RNA isolated from a mammalian mitochondria was investigated for Poly-A sequences. 34–39% of mitochondrial RNA was found to contain Poly-A as tested by two different techniques. The Poly-A sequences are 150–180 nucleotides (4–5S) long. The possibility of poly-A containing RNA being a cytoplasmic contaminant was excluded by treating mitochondria with RNAse and digitonin.

Differential effects of ethidium bromide on cytoplasmic and mitochondrial protein synthesis

Received 25 July 1974 1. Introduction The phenanthridine dye ethidium bromide (EB) is well-known for its ability to bind to DNA molecules. In eukaryotic cells, even at very low concentrations, EB is known to affect mitochondrial specific events [ 13. In mouse BHK cells 0.1 to 5 gg/ml EB specifically in- hibits mitochondrial DNA synthesis [2] . This drug is also known to inhibit mitochondrial specific RNA syn- thesis [ 1,3] , probably by binding to the DNA template thus interfering with chain initiation [4,5]. Consequent- ly EB has been widely used as a tool to study mitochon- drial specific transcription [6,7,8] . Isolated mitochondria from various cell types are known to incorporate both synthetic and natural poly- nucleotides which participate in intramitochondrial translation [9-l 21. Using an in vitro mitochondrial system from Xenopus, Grivell and Metz [ 121 recently demonstrated that EB directly interferes with the intramitochondrial phenylalanine incorporation di- rected by imported poly (U). This observation coin- cides with another indirect observation that EB may have an inhibitory effect on mitochondrial protein syn- thesis [ 131. We have verified these indirect results using a highly active in vitro protein synthesizing system from Ehrlich ascites mitochondria [ 141. Our results show that EB specifically inhibits mitochondrial but not cytoplasmic in vitro protein synthesis by prevent- ing polysome formation. 2. Materials and methods Ehrlich ascites hypotetraploid cells were grown for

A sensitive in vitro protein synthesizing system from ehrlich ascites mitochondria

Abstract The S-25 fraction prepared from digitonin washed mitochondria is highly active in poly(U) directed phenylanine incorporation when supplemented with t-RNA. Ribosomes prepared from the S-25 fraction contain 58S monomeric ribosomes and 40S and 29S subunits. Further, these ribosomes contain 21S and 13S rRNA. No detectable cytoplasmic specific ribosomal particles and also rRNA was detected in the mitochondrial S-25 preparation. Ribosomes from mitochondrial S-25 have specific requirement for mitochondrial specific supernatant factors for complete activity.

The abortive synthesis of new dna chains in ehrlich asc1tes tumour cells treated with nitrogen mustard (HN2)

Abstract Nitrogen mustard (HN2)- sensitive Ehrlich ascites tumour cells, exposed to HN2 in vivo , showed an inhibition of DNA synthesis which increased with dosage. The effects of alkylation involved at least three distinct components: ( 1 ) interference with new 9S DNA chain formation, ( 2 ) slowing of the rate of chain growth and ( 3 ) loss of newly formed short chains. The dominant effect seemed to be abortive synthesis of new 9S chains; this effect could account for most of the inhibition of DNA synthesis if an initial rapid synthesis of 9S DNA were accompanied by an initial rapid rate of destruction of these chains. By relating the known frequencies of guaninyl alkylations to the postulated ‘replicon’ size observed in control experiments, it appears that only difunctional alkylation frequencies can be directly correlated with the inhibition of discontinuous DNA synthesis by HN2, Mechanistically, this could reflect interference of di-guaninyl alkylations with the integration of 9S chains into 30S, 44S and higher molecular weight species of DNA by ligases. The resulting obstructed short chains, ≦ 9S, might be exposed and so be vulnerable to destruction by the increased nuclease activities expected after alkylation.

Phospholipid Stereospecificity in Liposomal Modulation of Nitrogen Mustard Action

Liposomes made of L(α) dipalmitoyl phosphatidyl choline (DPPC) enhance the antitumor efficacy of nitrogen mustard (HN2) in vivo, (Ritter et al, 1981) and in vitro (Ritter et al, 1987). Thus, liposomes act on the tumor cells directly rather than by altering NH2 distribution within the animal. In addition, since HN2 and DPPC liposomes do not associate, liposomes do not physically carry HN2 into tumor cells (Ritter et al, 1987). In lip-osome-treated cells though only slightly more HN2 is taken up, the amounts of cytolytic activity and DNA alkylation are doubled by comparison with HN2-treated controls (Ritter et al, 1987). Thus, liposomes increase the amount of HN2 distributed to the nucleus. By contrast, following Ca-H-treatment there is a 31% increase in cellular HN2 uptake but 50% decrease in DNA alkylation and cytolysis (Ritter et al, 1987). The effects on uptake seem to be much less important than the effects on intracellular distribution.