Hyperthermic modification of cyclophosphamide metabolism in rat hepatic microsomes and liver slices

The ability of rat liver microsomes and liver slices to metabolize the antineoplastic compound cyclophosphamide was studied at 37° and at elevated temperatures comparable to those used for human systemic hyperthermic antineoplastic therapy. Temperatures above 40.5° and 41.8° inhibited cyclophospamide metabolism by microsomes and liver slices respectively. Therefore, cyclophosphamide may not be a suitable drug for combination with systemic hyperthermia in cancer therapy.     

Habitat assortment of sexes and water balance in a dioecious grass

Summary Eggs of the carabid beetle Pterostichus oblongopunctatus kept under optimum conditions in petri dishes had a mean mortality of 8% (n=200), while the mortality of eggs incubated in fresh litter was 83% (n=250). Heating the litter up to 70° C and thus eliminating other organisms from the litter fauna reduced mortality to 18% (n=250). When eggs were deposited by the females themselves in a perspex cylinder containing an undisturbed soil sample, the mean egg mortality was 70% (n235), but when predators had been eliminated the mortality was only 7% (n235). The assumption of Grüm (1975) that egg mortality can be neglected does not hold for P. oblongopunctatus. The predators of the eggs must be common species, but it is not yet known to which family they belong.     

A DNA-binding fraction of mouse kidney 3-ketosteroid reductase: Comparison with androgen receptors

Since approximately 1% of 3-ketosteroid reductase (which metabolizes dihydrotestosterone [17β-hydroxy-5α-androstan-3-one] to 5α-androstane-3α,17β-diol or 5α-androstane-3α,17β-diol) from mouse kidney cytosol adheres to DNA under conditions that allow virtually complete androgen receptor binding, these two DNA-binding activities were compared in cytosol extracts of mouse kidney and hypothalamus-preoptic area. This DNA-binding fraction of 3-ketosteroid reductase was distinguished from androgen receptor in several ways: (1) its pattern of elution from DNA-cellulose with steps of increasing NaC1 concentration differed from that for receptors from wild-type kidney; (2) it was influenced differently by the mutation Tfm, both in level and in DNA-cellulose elution pattern; (3) in mouse kidney cytosol it was relatively stable at moderate (25°C) temperatures which rapidly inactivated ligand-free androgen receptors in the same cytosols; (4) the DNA-binding was not proportional to androgen receptor levels between two wild-type tissues, the hypothalamus-preoptic area and kidney. By these criteria, a simple relationship of androgen receptors and a DNA-binding fraction of 3-ketosteroid reductase activity is unlikely.     

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.     

Properties of receptors for epidermal growth factor in detergent solution: Evidence for heterogeneous aggregated states

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal forece and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for ¹²⁵I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with ¹²⁵I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble and -insoluble fractions. Gel chromatography of the detergent-soluble membrane fraction on Sepharose 6-B revealed heterogeneity of ¹²⁵I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Stokes radius of 95 Å. Operationally soluble ¹²⁵I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of ¹²⁵I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

Macrophage migration stimulation factor, migration inhibition factor and the role of suppressor cells in their production.

Guinea pig lymph node lymphocytes and human peripheral blood lymphocytes when stimulated by specific antigen or mitogen will release factors that affect in vitro macrophage migration. Migration inhibition factor production appears to be under the control of suppressor cells which are T lymphocytes. When suppressor cells are generated by stimulation with Con A for 4 days, migration stimulation factor (M.St.F.) activity is found. In other situations where M.St.F. is found this is thought to be due to increased suppressor cell activity. For example, young adults produce this lymphokine when stimulated with Con A, whereas aged individuals produce MIF. Concanavalin A appears to be the mitogen of choice for M.St.F. production, and phytohemagglutinin for MIF production. The release of this putative factor M.St.F. from suppressor T cells helps to explain some of the difficulties that have existed in studies of macrophage migration inhibition.     

Subcellular distribution of the different platelet proteins phosphorylated on exposure of intact platelets to ionophore A23187 or to prostaglandin E1. Possible role of a membrane phosphopolypeptide in the regulation of calcium-ion transport.

Exposure of 32P-labelled human platelets to ionophore A23187 results in an increased incorporation of 32P into polypeptides with apparent mol.wts. of 47 000 (P47) and 20 000 (P20), whereas exposure to prostaglandin E1 results in increased labelling of polypeptides with apparent mol.wts. of 24 000 (P24) and 22 000 (P22) [Haslam, Lynham & Fox (1979) Biochem. J. 178, 397-406]. Labelled platelets that had been incubated with ionophore A23187 or prostaglandin E1 were sonicated and rapidly separated into three fractions by differential centrifugation. Electron microscopy and measurement of marker enzymes indicated that the 1300-19 000 gav. particulate fraction was enriched in granules, mitochondria and plasma membranes, that the 19 000-90 000 gav. particulate fraction was enriched in both intracellular and plasma membranes and that the 90 000 gav. supernatant contained only soluble proteins. 32P-labelled phosphopolypeptide P47 was present almost exclusively in the 90 000 gav. supernatant, whereas phosphopolypeptide P20 was largely dephosphorylated under fractionation conditions that protected other phosphopolypeptides. 32P-labelled phosphopolypeptide P24 was enriched in both particulate fractions, but particularly in the 19 000-90 000 gav. fraction, and may therefore be present in both the intracellular and plasma membranes. Phosphopolypeptide P22 appeared to be similarly distributed. Both particulate fractions were capable of the ATP-dependent oxalate-stimulated uptake of Ca2+. When the 19 000-90 000 gav. membrane fraction was prepared from platelets that had been incubated with ionophore A23187, active uptake of Ca2+ did not occur, but when this fraction was isolated from platelets that had been exposed to prostaglandin E1, uptake of Ca2+ was significantly greater than observed with the corresponding membranes from control platelets. It is suggested that phosphorylation of polypeptide P24 (or P22) by a cyclic AMP-dependent protein kinase may promote the active transport of Ca2+ out of the platelet cytosol.     

SDS removal from protein by polystyrene beads

The applications of sodium dodecyl sulfate (SDS) to molecular weight determination (1,2) and for the separation of protein subunits (3) have been of immense value in biochemical studies [see Waehneldt (4) for a general review]. The tight stoichiometric binding of SDS to polypeptide chains has proven to be a nuisance if one desires to recover the activity of the isolated polypeptides. Removal of the SDS has been affected by the use of anion exchangers in the presence (5,6) and absence of urea (6). However, the residual levels of SDS or urea are often quite unsatisfactory for further protein studies.We have attempted to adapt the procedure of Holloway for the removal of Triton X-100 by Bio-Beads to the removal of SDS. We chose bovine serum albumin as a test protein since it has well-established strong binding properties for linear-chain fatty acids (7).     

The interaction of dianhydrogalactitol with DNA in cultured Yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand cross-linking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-linked.