Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells

The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear. We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells. CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance. SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM. CsA and SDZ 33-243 at 10 microM increased [3H]vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively. At 10 microM, these compounds also increased [3H]VCR accumulation by 3.5- and 4.0-fold, respectively. [3H]VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively. Kinetic analysis of [3H]VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity [3H]VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM. In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM. These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein.     

Effect of coexposure to 50 Hz magnetic fields and an aneugen on human lymphocytes,determined by the cytokinesis block micronucleus assay

Interference of 50 Hz extremely low frequency magnetic fields (ELF-MF) with the known aneugen vinblastine (VBL) on micronucleus formation was tested with the in vitro cytokinesis block micronucleus assay in human lymphocyte cultures. Isolated lymphocyte cultures were prepared from 18 individuals. Three groups of quadruplicate cultures from six unrelated individuals were exposed to 50 Hz ELF-MF of background (bkg), 80 and 800 microT, respectively, during the complete incubation period (72 h). Twenty-four hours after culture initiation, one replicate culture from each individual within each ELF-MF group was exposed to 0, 5, 10, or 15 ng/ml VBL. The isolated lymphocyte cultures were scored for the presence of micronuclei, the nuclear division index (NDI), and apoptosis. As expected, increased VBL concentration resulted in an increased micronucleus and apoptosis frequency and in a decreased NDI. In the presence of VBL, there was a systematic tendency for increased micronucleus and apoptosis frequency in the ELF-MF exposed groups compared to the bkg group. In the absence of VBL, we observed no statistically significant effect of ELF-MF on micronucleus induction or apoptosis frequency, but the NDI was significantly higher in the 800 microT group compared to the other groups, suggesting an effect of ELF-MF on cell proliferation. An interaction between ELF-MF and VBL on NDI was observed. This interaction reflected the drastic decrease in NDI due to coexposure to VBL.     

CELL KINETIC ALTERATIONS IN NORMAL AND NEOPLASTIC CELL POPULATIONS IN VITRO AND IN VIVO FOLLOWING VINCRISTINE*. A REPLY TO DR CAMPLEJOHN'S REVIEW ARTICLE†

It is shown that the lethal action of vincristine (VCR) is dose-dependent and may occur at interphase and mitosis. In general, the VCR dose used to destroy cells must be approximately ten times higher than that used to arrest cells in mitosis at metaphase. There is strong evidence that cells can survive metaphase arrest by a sublethal dose of VCR either completing cytokinesis normally after metabolism of the drug or becoming polyploid because of an impaired mitotic spindle apparatus. These cells are not doomed to die, at least in some cell systems. Furthermore, there is strong evidence in three animal tumour systems (transplantable and autochthonous tumours) that VCR is able to induce in vivo partial synchronization of proliferating tumour cells and/or recruitment of resting cells into the proliferating compartment. Failures to induce partial synchrony in cell populations by VCR may be attributed to resistance to VCR or cytolysis or slow proliferation of cells in badly vascularized tumours. Chemotherapy after synchronization seems to be effective as shown by non-randomized trials in bad-risk patients with solid tumours and acute leukaemias. In a randomized co-operative trial results of the two-drug synchronization protocol in patients with non-Hodgkin's lymphoma of high grade malignancy were statistically better than those of a four-drug protocol (COPP) established empirically. The two-drug protocol was equally effective as the four-drug protocol in Hodgkin's disease. Side-effects were less pronounced with the so-called synchronization scheme.     

The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor

We have defined the structure of the Osteoclast Functional Antigen (OFA) by immunological and biochemical means. OFA is an abundant surface antigen in human and animal osteoclasts and has been characterized previously by monoclonal antibodies 13C2 and 23C6, one of which mimicks the inhibitory activity of calcitonin on osteoclastic bone resorption. By the following criteria we show that OFA is a member of the integrin family of extracellular matrix receptors and is identical, or at least highly related, to the vitronectin receptor (VNR) previously isolated from placenta and melanoma cells. Immunoprecipitation analysis demonstrates that OFA from osteoclasts and a monkey kidney cell line Vero is a heterodimeric molecule of 140 kD (alpha chain) and 85 kD (beta chain) under nonreducing conditions; on reduction at least one low molecular mass (alpha') species (of approximately 30-kD size) is released, resulting in a 120/100-kD dimer. Immunoblots of OFA isolated from osteoclasts and Vero cells and VNR purified from placenta and probed with heterosera to OFA and monoclonal antibodies to platelet gp111a (VNR beta chain) show immunological cross- reactivity between the alpha chains of OFA and VNR and the use of gp111a as a beta chain by both. OFA from Vero cells binds to an Arg-Gly- Asp containing peptide (GRGDSPPK) isolating a heterodimer recognized by anti-OFA monoclonal antibodies, 13C2 and 23C6. Immunohistochemical analysis showed a similar tissue distribution in humans for the antigen recognized by anti-OFA antibodies, a monoclonal antibody, LM142, raised to melanoma VNR, polyclonal antibodies to the placental VNR and a monoclonal antibody to the presumptive VNR beta chain, platelet glycoprotein 111a. Finally, NH2 terminal amino acid sequencing showed that the amino-terminus of the monkey alpha chain was identical in the 12 assigned residues to that of human VNR alpha chain. The beta chain sequence of OFA differed at least 1 (and up to 4) positions from platelet gp111a (VNR beta) in the first 18 amino acids sequenced. These, and other, data provide the first indication of a function for the VNR and suggest that cell-cell and cell-extracellular matrix interactions involving integrins may play an important role in bone physiology.     

Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.     

Natural and synthetic forms of insulin-like growth factor-1 (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding

Insulin-like growth factor-1 (IGF-1), whether recombinant, chemically-synthesised or purified from bovine colostrum, was equipotent in radioreceptor assays with IGF-1 or insulin-like growth factor-2 (IGF-2) as radioligand as well as in its ability to stimulate protein synthesis in L6 myoblasts. The N-terminal truncated, destripeptide derivative of IGF-1 was approximately 7 times more potent than IGF-1 in the protein synthesis bioassay. This increased activity occurred equally with the peptide purified from bovine colostrum as with chemically-synthesised material. The higher potency of the truncated form was not associated with an increased ability to compete for IGF-1 binding to L6 myoblasts.     

14-Dehydro-19-nortestosterone and its 7 -methyl derivative

14-Dehydro-19-nortestosterone and its 7α-methyl derivative were synthesized. The former was found to be approximately 100 and the latter 1000 times as active as testosterone in chick comb (local application) assays. In rat assays (subcutaneous), 14-dehydro-19-nortestosterone was approximately one-half as active as, or equal to testosterone in the ventral prostate or levator ani assays respectively, whereas its 7α-methyl derivative still retained its high potency (100 times as active as testosterone) in either type of assay.     

Reaction-kinetic parameters of glycidamide as determinants of mutagenic potency

Values for reaction-kinetic parameters of electrophiles can be used to predict mutagenic potency. One approach employs the Swain-Scott relationship for comparative kinetic studies of electrophilic agents reacting with nucleophiles. In this way glycidamide (GA), the putatively mutagenic/carcinogenic metabolite of acrylamide, was assessed by determining the rates of reaction with different nucleophiles. The rate constants (kNu) were determined using the "supernucleophile" cob(I)alamin [Cbl(I)] as an analytical tool. The Swain-Scott parameters for GA were compared with those of ethylene oxide (EO). The substrate constants, s values, for GA and for EO were found to be 1.0 and 0.93, respectively. The reaction rates at low values of nucleophilic strength (n=1-3), corresponding to oxygens in DNA, were determined to be 2-3.5 times higher for GA compared to EO. GA was also more reactive than EO towards other nucleophiles (n=0-6.4). The mutagenic potency of GA was determined in Chinese hamster ovary cells (hprt mutations in CHO-AA8 cells per dose unit with gamma-radiation as reference standard). The potency of GA was estimated to be about three mutations per 10(5) cells and mMh corresponding to about 40 rad-equ./mMh. A preliminary comparison of the mutagenic potency (per mMh and as rad-equivalents) of GA and EO shows an approximately seven times higher potency for GA. A higher mutagenic potency of GA compared to EO is compatible with expectation from reaction-kinetic data of the two compounds. The data confirmed that GA is not a strong mutagen, which is in line with what is expected for simple oxiranes. The present study shows the value of cob(I)alamin for the determination of reaction-kinetic parameters and their use for prediction of mutagenic potency.     

Labeling of integrin alpha v beta 3 with 58Co(III). Evidence of metal ion coordination sphere involvement in ligand binding.

Integrin-mediated cell adhesion to the extracellular matrix is divalent metal ion-dependent; however, a demonstration of the interaction between native integrins and divalent metal ions is lacking. Here we provide direct evidence that the vitronectin receptor (VNR) is a metalloprotein. The unique electron shell of Co(II), an ion which we show supports ligand recognition by VNR, enables its oxidative conversion to inert Co(III). This property facilitated "affinity labeling" of VNR with 58Co(III) by oxidation of the metal ion in situ (i.e. in position). An average of 3.5 +/- 0.5 mol of cobalt were incorporated per mol of VNR. The ability of VNR to bind metal ions was independently confirmed by examining the interaction between VNR and Mn2+ under native conditions. The apparent high affinity between VNR and Mn2+ allowed us to observe the specific binding between 54Mn2+ and VNR by equilibrium gel filtration studies. Interestingly, the oxidative incorporation of Co(III) into VNR specifically blocked ligand binding, suggesting that the coordination sphere of metal ion bound to VNR is a critical determinant in integrin-ligand recognition. Furthermore, Mn2+ abolished the oxidative affinity labeling of VNR with Co(III) and consequently blocked the inactivation of VNR by in situ incorporation of Co(III). Thus, Mn2+ and Co2+ bind to the same or mutually exclusive sites on VNR. These observations provide the first demonstration that an integrin, specifically VNR, is a metalloprotein and demonstrate a functional link between the coordination sphere of the bound metal ion and ligand recognition by this receptor.     

Novel Bioactivities from a Coral, Galaxea fascicularis: DNase-like Activity and Apoptotic Activity Against a Multiple-Drug-Resistant Leukemia Cell Line

From the coral Galaxea fascicularis, a crude mucus-like extract (MS) and subsequently its purified component (P6) appear to contain a DNase-like activity that indiscriminately digested λDNA, as well as naked genomic DNAs isolated from a multiple-drug-resistant murine leukemia cell line, P388/VCR, and a nontransformed liver cell line, BL8L. However, MS and P6 specifically induced in situ DNA digestion in cultured P388/VCR cells from 30 minutes onward. After 3 days of incubation with MS or P6, DNA degradation coincided with complete killing of P388/VCR. In situ fluorescent labeling of fragmented DNA revealed that P6 induced apoptosis of P388/VCR cells, occurring as early at 1.5 hours. By day 3, all the P6-treated leukemia cells were apoptotic. In contrast, P6 caused neither in situ DNA digestion, nor apoptosis in the untransformed BL8L cells. Whether the DNase-like action of P6 is independent of or responsible for triggering the intrinsic endonuclease activity in the leukemia cell, thus leading to apoptosis, remains an object for further research. Nevertheless, the specificity of the apoptotic action of P6 on P388/VCR cells indicates its potential role in the development of an anticancer agent. Received July 6, 1998; accepted December 21, 1998     

Staurosporine, a potent inhibitor of C-kinase, enhances drug accumulation in multidrug-resistant cells

Staurosporine, a potent inhibitor of C-kinase, enhances accumulation of vincristine (VCR) in multidrug-resistant cells. We investigated this enhancement by two methods: (I) ATP-dependent VCR binding system; (II) azidopine photolabeling system. The ATP-dependent VCR binding to the resistant cell membrane was inhibited more efficiently by staurosporine than by verapamil. Staurosporine also inhibited the azidopine photolabeling of P-glycoprotein. These results indicate that staurosporine, an inhibitor of C-kinase, might directly bind to P-glycoprotein as well as antitumor agents and Ca2+ channel blockers. These findings also indicate that C-kinase might be involved in the function of P-glycoprotein.     

Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.     

Beneficial effects of quercetin on vincristine-induced liver injury in rats: Modulating the levels of Nrf2/HO-1, NF-kB/STAT3, and SIRT1/PGC-1α

Our experimental objective was to investigate the hepatotoxic effect of vincristine (VCR) administration in rats and determined whether combined therapy with Quercetin (Quer) ensured protection. Five groups with seven rats each were used for this purpose, and experimental groups were formulated as follows: Control group; Quer group; VCR group; VCR plus Quer 25 group; VCR plus Quer 50 group. The results showed that VCR significantly increased the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes. Besides, VCR caused considerable increases in the malondialdehyde (MDA) contents, along with significant decreases in reduced glutathione levels, superoxide dismutase, catalase, and glutathione peroxidase enzyme activities in the rat livers. Quer treatment in VCR toxicity markedly decreased the activity of ALT, AST, ALP enzymes, and MDA contents and enhanced the activities of antioxidant enzymes. The results also showed that VCR significantly increased the levels of NF-kB, STAT3, and the expression of caspase 3, Bax, and MAP LC3 and decreased the expression of Bcl2 and levels of Nrf2, HO-1, SIRT1, and PGC-1α. Compared to the VCR group, Quer treatment exhibited significantly lower levels of NF-kB, STAT3, and the expression of caspase 3, Bax, and MAP LC3, and higher levels of Nrf2, HO-1, SIRT1, and PGC-1α. In conclusion, our study demonstrated that Quer could alleviate the harmful effects of VCR via activation of NRf2/HO-1 and SIRT1/PGC-1α pathways, and via attenuation of oxidative stress, apoptosis, autophagy, and NF-kB/STAT3 pathways.     

Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

Bisbibenzyl derivatives sensitize vincristine-resistant KB/VCR cells to chemotherapeutic agents by retarding P-gp activity

P-glycoprotein (P-gp) is known to mediate multidrug resistance (MDR) by acting as an efflux pump to actively transport chemotherapeutic agents out of carcinoma cells. Inhibition of P-gp function may represent one of the strategies to reverse MDR. We have previously reported that marchantin C (MC), a macrocyclic bisbibenzyl compound from liverworts, exerts anti-tumor activity as an antimitotic agent. This study was designed to evaluate the possible modulatory effect of MC and its three synthetic derivatives (MC1, MC2 and MC3) on P-gp in VCR-resistant KB/VCR cells. Results of the cytotoxicity assay revealed that MC was the most potent inhibitor of cell proliferation in both KB and KB/VCR cells among these four compounds, while the three MC-derived chemicals had little anti-proliferative activity under the same condition. However, in P-gp-expressing MDR cells, analysis of potency of these compounds in enhancing cytotoxicity of VCR led to the identification of MC2 as a more effective chemical on reversal of resistance. Further study showed that MC2 was able to reduce efflux of rhodamine-123, and in turn, increase the accumulation of rhodamine-123 and adriamycin in KB/VCR cells, indicating that MC2 re-sensitized cells to VCR by inhibition of the P-gp transport activity. In addition, the combination of MC2 and VCR at a concentration that does not inhibit cell growth resulted in an induction of apoptosis in KB/VCR cells. These results suggest that MC2, as a novel and effective inhibitor of P-gp, may find potential application as an adjunctive agent with conventional chemotherapeutic drugs to reverse MDR in P-gp overexpressing cancer cells.     

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.     

Hypoxia-selective radiosensitization of mammalian cells by nitracrine, an electron-affinic DNA intercalator

The radiosensitizing ability of the 1-nitroacridine nitracrine (NC) is of interest since it is an example of a DNA intercalating agent with an electron-affinic nitro group. NC radiosensitization was evaluated in Chinese hamster ovary cell (AA8) cultures at 4 degrees C in order to suppress the rapid metabolism and potent cytotoxicity of the drug. Under hypoxic conditions, submicromolar concentrations of NC resulted in sensitization (SER = 1.6 at 1 mumol dm-3). Sensitization was also seen under aerobic conditions but a concentration more than 10-fold higher was required. In aerobic cultures NC radiosensitization was independent of whether cells were exposed before and during, or after, irradiation. Postirradiation sensitization was not observed under hypoxic conditions. The time dependence of NC uptake and the development of radiosensitization were similar (maximal at 30 min at 4 degrees C under hypoxia) suggesting that sensitization, unlike cytotoxicity, is due to unmetabolized drug. NC is about 1700 times more potent as a radiosensitizer than misonidazole. This high potency is adequately accounted for by the electron affinity of NC (E(1) value at pH7 of -275 mV versus NHE) and by its accumulation in cells to give intracellular concentrations approximately 30 times greater than in the medium. However, concentrations of free NC appear to be low in AA8 cells, presumably because of DNA binding. If radiosensitization by NC is due to bound rather than free drug, it suggests that intercalated NC can interact very efficiently with DNA target radicals. This is despite a binding ratio in the cell estimated as less than 1 NC molecule/400 base pairs under conditions providing efficient sensitization. This work suggests a new approach in the search for more effective clinical radiosensitizers, and poses questions on the means by which intercalated drugs can interact with DNA damage.     

A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.     

Comparative behavioral pharmacology and toxicology of cocaine and its ethanol-derived metabolite, cocaine ethyl-ester (cocaethylene).

The present study compared the behavioral and toxic effects of cocaine and its ethanol derived metabolite, cocaine ethyl-ester (cocaethylene). Both drugs produced qualitatively similar psychomotor stimulant effects. Cocaine and cocaethylene increased locomotor activity in mice, with cocaine approximately four times more potent than cocaethylene. The durations of action of ED75 doses of each of the drugs were comparable. Each of the drugs also produced stimulation of operant responding in rats. In rats and squirrel monkeys trained to discriminate cocaine injections from saline, cocaine was approximately three to five times more potent than cocaethylene in producing these cocaine-like interoceptive effects. In contrast to the behavioral effects, cocaine and cocaethylene were equipotent in producing convulsions, and cocaethylene was more potent than cocaine in producing lethality. These results suggest that the conversion of cocaine to cocaethylene with simultaneous cocaine and alcohol use may produce an increased risk of toxicity due to a decrease in the potency of cocaethylene in producing psychomotor stimulant effects, and its increased potency in producing toxicity.