Physiology and endocrinology of zinc accumulation during the female squirrelfish reproductive cycle.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of hepatic zinc than any other studied animal. This accumulation is accompanied by high expression of the zinc-binding protein, metallothionein (MT), and is strongly correlated to the onset of sexual maturity. In an attempt to further characterize the timeframe of this accumulation, and to possibly discern any potential mediators, we examined the physiology and endocrinology of the yearly reproductive cycle of mature female squirrelfish. There are two separate reproductive events during the year in December-January and again in March-April, as evidenced by peaks in ovarian growth, VTG production, steroid levels, zinc accumulation and redistribution. Increased hepatic zinc seems to be preceded by a necessary increase in MT, but this was not clearly correlated to plasma 17beta-estradiol, testosterone, or progesterone levels. The plasma zinc protein vitellogenin (VTG) is one, but probably not the predominant, vehicle for the transport of hepatic zinc to the ovary.

Effects of 17 beta-estradiol on levels and distribution of metallothionein and zinc in squirrelfish.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.

Evaluation of the teicoplanin broth microdilution and disk diffusion susceptibility tests and recommended interpretive criteria.

Comparative teicoplanin in vitro susceptibility data were generated for 1201 Gram-positive US clinical trial isolates using standardized broth microdilution and disk diffusion techniques. Based on the results of this study, the following interpretive criteria for teicoplanin are recommended: for MIC tests, less than or equal to 8 micrograms/ml = susceptible, 16 micrograms/ml = moderately susceptible, and greater than or equal to 32 micrograms/ml = resistant; and for disk (30 micrograms) tests, greater than or equal to 14 mm = susceptible, 11-13 mm = intermediate, and less than or equal to 10 mm = resistant.

In vitro and in vivo candicidal activities of 2-(p-n-hexylphenylamino)-1,3-thiazoline.

The 2-amino-1,3-thiazoline, 2-(p-n-hexylphenylamino)-1,3-thiazoline (MDL 20,245) killed 10(5) logarithmic or stationary phase Candida albicans/ml in less than 1 h. Miconazole killed logarithmic phase cells at that rate, but miconazole, clotrimazole or econazole killed stationary phase cells at a slower rate of 10(2)-10(4) cells/ml in 24 h. MDL 20,245 induced efflux of K+ and L[U-14C] lysine from C. albicans, indicating that the candicidal mechanism is to exert direct damage upon the cytoplasmic membrane. The activity of MDL 20,245 in vitro was antagonized by fatty acids, triglycerides and phospholipids. Topical application of MDL 20,245 ointment (10% w/v) twice per day for 4 days to rats suppressed C. albicans-induced vaginitis 100%. Single-dose regimens of MDL 20,245, miconazole or clotrimazole correlated with 97, 90 and 73% suppression, respectively. These data suggest that MDL 20,245 may be effective in the treatment of C. albicans-induced vaginitis in humans.

Potentiation of antitumor and antimetastatic activities of alpha-difluoromethylornithine by interferon inducers.

The objective of the present investigation was to study the potentiation of antitumor and antimetastatic activities of DL-alpha-difluoromethylornithine (DFMO) by inducers of interferon, namely, tilorone and polyriboinosinic:polyribocytidilic acid complex [poly(l) X poly(C)]. The results of this study indicate that these interferon inducers enhance the antitumor activity of DFMO against B16 melanoma and Lewis lung carcinoma in mice. In B16 melanoma, DFMO, tilorone, or poly(l) X poly(C), when administered alone, showed 85, 39, and 39% of inhibition of tumor growth, respectively. However, a combination of DFMO and tilorone or poly(l) X poly(C) resulted in 98 and 95% inhibition of growth, with about 20% of animals showing no detectable tumors. This potentiation appears to be related to the ability of the compounds to induce interferon, since an analogue of tilorone, MDL 10,842, neither induced interferon nor potentiated the antitumor activity of DFMO. The data also indicate that this combination is particularly effective when the tumor burden is relatively low. When tilorone was given 7 days after tumor inoculation, it did not show any potentiation of antitumor activity of DFMO. The studies with Lewis lung carcinoma also showed that the interferon inducers potentiated both the antitumor and antimetastatic activities of DFMO. DFMO or tilorone administered alone showed 28 and 46% inhibition of tumor growth and 80 and 58% inhibition of metastases, respectively. Poly(l) X poly(C) by itself did not have any effect on the tumor growth and metastases. However, a combination of DFMO and tilorone brought about 78% inhibition of tumor growth and 99.5% inhibition of metastases, with 87% of the animals free of any metastases. A combination of DFMO and poly(l) X poly(C) also showed a potentiation of both antitumor activity (58% inhibition) and antimetastatic activity (94% inhibition), with 62% of the animals free of any detectable metastases. The mechanism underlying this tumor suppression by combination of DFMO and interferon inducers is not yet known. Enhancement of host immune response or interferon-mediated cytotoxicity could account for the observed marked suppression of tumor growth. Previous studies using interferon and the data reported here with interferon inducers, along with the relatively nontoxic nature of DFMO, suggest a potential use for the inhibitors of polyamine biosynthesis in combination with interferon or interferon inducers in cancer chemotherapy and other proliferative states.

Tumor suppression with a combination of alpha-difluoromethyl ornithine and interferon.

alpha-Difluoromethyl ornithine and mouse type 1 interferon, when administered simultaneously, were highly toxic to B16 melanoma cells in culture. Oral administration of alpha-difluoromethyl ornithine suppressed B16 melanoma development in mice 85 percent whereas interferon given subcutaneously inhibited tumor growth only 24 percent. Total or near total suppression of tumor growth was observed in mice receiving both treatments.

RMI 15,731 (1-[5-tetradecyloxy-2-furanyl]-ethanone), a new antirhinovirus compound.

RMI 15,731 (1-[5-tetradecyloxy-2-furanyl]-ethanone) is a new antiviral compound with activity specific for rhinoviruses. Virus synthesis in R-HeLa cells was susceptible to low concentrations (0.25 micrograms/ml) of the compound. RMI 15,731 was effective if added for 1 h before infection and removed or if added as late as 6 h postinfection. Low concentrations of the compound directly inactivated infectious rhinovirus in a time- and temperature-dependent fashion. RMI 15,731 appears to be a promising new antirhinovirus compound for clinical evaluation since it (i) blocks intracellular virus synthesis (without cellular toxicity), and (ii) directly inactivates virions. The last property may be important in limiting virus shedding from an infected host.

Bis-basic-substituted polycyclic aromatic compounds. A new class of antiviral agents. 8. Bis-basic derivatives of carbazole, dibenzofuran, and dibenzothiophene.

A series of bisalkamine esters, bis-basic ethers, and bis-basic ketones of carbazole, N-ethylcarbazole, dibenzofuran, and dibenzothiophene was synthesized and evaluated for antiviral activity. The series also included two bis-basic alkanes of N-ethylcarbazole and one bis-basic carboxamide of dibenzofuran. Structure-activity relationships indicated that within the carbazole and N-ethylcarbazole series the bisalkamine esters gave the most active derivatives while the bis-basic ketone derivatives of dibenzofuran and dibenzothiophene afforded the more potent compounds within the respective series. The [6,5,6]heterocyclic nuclei were compared with the [6,5,6] aromatic nuclei (fluroene and fluoren-9-one) including tilorone with respect to antiviral activity against encephalomyocarditis (EMC) virus. Maximum activity was associated with the bis-basic ketone side chain and fluoren-9-one nucleus.

Bis-basic-substituted polycyclic aromatic compounds. A new class of antiviral agents. 7. Bisalkamine esters of 9-oxoxanthene-2,7-dicarboxylic acid, 3,6-bis-basic ethers of xanthen-9-one, and 2,7-bis(aminoacyl)xanthen-9-ones-xanthenes, and -thioxanthenes.

3,6-Bis[2-(dimethylamino)ethoxy]-9H-xanthen-9-one dihydrochloride (4, RMI 10874DA) and 1,1'-(9H-xanthene 2,7-diyl)bis[2-(dimethylamino)ethanone] dihydrochloride (16, RMI 11513DA) were found to prolong survival of mice infected with lethal challenges of encephalomyocarditis (EMC) virus. They were effective by oral as well as subcutaneous administration and showed broad-spectrum antiviral activity. They were selected for preclinical evaluation from the five series of compounds named in the title that were synthesized in analogy to tilorone and related fluorenone derivatives, described earlier. In addition to 4 and 16, compounds 11, 12, 17, and 18 showed high antiviral activity on oral as well as subcutaneous administration. High antiviral activity on subcutaneous admistration was found in the bisalkamine esters 1,2, and 14, the bis(aminoacyl)xanthenes 23 and 26, the bis(aminoalkylene)xanthene 31, the bis(aminoacyl)thioxanthenes 34-40, and the bis-basic ethers of 9-benzylide-nexanthenes 41 and 42. Structure-activity relationships showed a decrease of oral activity with increased length of side chains and increased molecular weight of dialkylamino substituents of 3,6-bis-basic ethers of xanthen-9-one and of 2,7-bis(aminoacyl)xanthenes and-xanthen-9-ones. At least one carbonyl or alkenyl function in conjugation to the xanthene nucleus either at the 9 position of the nucleus or in the side chains is required for high antiviral activity.

Enhancement of interferon production in vitro: a property of tilorone-poly rL:rC/DEAE-Dextran.

Tilorone and Poly rI:rC, in the presence of DEAE-dextran, were found to exhibit a marked synergism with respect to the induction of interferon in L929 and primary mouse embryo fibroblasts, but not human foreskin fibroblasts, in cell cultures. The degree of synergism was proportional to the concentrations of tilorone and Poly rI:rC and was influenced by the times of addition of the compounds relative to each other.

Tilorone hydrochloride: an orally active antiviral agent.

Tilorone hydrochloride, 2,7-bis[2-(diethylamino)ethoxy]fluoren-9-one dihydrochloride, given orally to mice before they are infected is active against at least nine viruses of both RNA and DNA groups. The compound is effective when given prophylactically; the optimum time of treatment depends on the route of infection.

Tilorone hydrochloride: mode of action.

An antiviral serum component is found in mice treated orally with tilorone hydrochloride. The active material fulfills sufficient biological criteria to be classified as an interferon.