GnRHa-mediated stimulation of the reproductive endocrine axis in captive Atlantic bluefin tuna, Thunnus thynnus.

A controlled-release implant loaded with GnRH agonist (GnRHa) was used to induce spawning in Atlantic bluefin tuna (Thunnus thynnus) during two consecutive reproductive seasons. The fish were implanted underwater and sampled between days 2 and 8 after treatment. At the time of GnRHa treatment, females were in full vitellogenesis and males in spermiation. There was a rapid burst of pituitary luteinizing hormone (LH) release at day 2 after treatment in GnRHa-treated fish, and circulating LH remained elevated up to day 8 after treatment. In contrast, control fish had significantly lower levels in the plasma, but higher LH content in the pituitary, as observed in many other cultured fishes that fail to undergo oocyte maturation, ovulation and spawning unless induced by an exogenous GnRHa. Plasma testosterone (T) and 17ß-estradiol (E(2)) were elevated in response to the GnRHa treatment in females, while 11-ketotestosterone (11-KT) but not T was elevated in males. Even though oocyte maturation and ovulation did occur in GnRHa-induced fish, no significant elevations in 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) or 17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S), in either the free, conjugated or 5ß-reduced,3α-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20ß-P levels occurred in both males and females at day 8 after treatment. Histological sections of the ovaries in these females contained oocytes at the migrating germinal vesicle stage, suggesting the role of this hormone as a maturation-inducing steroid in Atlantic bluefin tuna. In conclusion, the GnRHa implants activated effectively the reproductive endocrine axis in captive Atlantic bluefin tuna broodstocks, through stimulation of sustained elevations in plasma LH, which in turn evoked the synthesis and secretion of the relevant sex steroids leading to gamete maturation and release.

Activity of the antimicrobial polypeptide piscidin 2 against fish ectoparasites.

Abstract The antiparasitic effects of piscidin 2, an antimicrobial polypeptide (AMPP) first isolated from mast cells of hybrid striped bass, were tested against three protistan ectoparasites of marine fish (the ciliates Cryptocaryon irritans and Trichodina sp., and the dinoflagellate Amyloodinium ocellatum) and one ciliate ectoparasite of freshwater fish (Ichthyophthirius multifiliis). I. multifiliis was the most susceptible parasite, with all theronts killed at 6.3 microg mL(-1) piscidin 2. The most resistant parasite was Trichodina, where a few cells were killed at 12.5 microg mL(-1), but several were still alive even at 100 microg mL(-1). C. irritans was of intermediate sensitivity, with some theronts killed at 12.5 microg mL(-1) and all killed at 25 microg mL(-1). High parasite density apparently exhausted the piscidin 2 before it could attain its maximal effect, but surviving parasites were often visibly damaged. The lower efficacy of piscidin 2 against marine parasites compared with the freshwater ciliate might be related to the inhibitory effects of high sea water cation levels. The tissue concentration of piscidins estimated in healthy hybrid striped bass gill (40 microg mL(-1)) suggests that piscidin 2 is lethal to the parasites tested at physiological concentrations and is thus an important component of innate defence in fish expressing this type of AMPP.

2-benzoxazolyl and 2-benzimidazolyl hydrazones derived from 2-acetylpyridine: a novel class of antitumor agents.

Here we describe the effects of novel benzoxazol-2-yl and benzimidazol-2-yl hydrazones derived from 2-pyridinecarbaldehyde and 2-acetylpyridine. The IC(50) values for inhibition of cell proliferation in KB-3-1, CCRF-CEM, Burkitt's lymphoma, HT-29, HeLa, ZR-75 and MEXF276L by most of the novel compounds are in the nanomolar range. In colony-forming assays with human tumor xenografts the compounds 2-actylpyridine benzoxazol-2-ylhydrazone (EPH52), 2-acetylpyridine benzoimidazol-2-ylhydrazone (EPH61) and 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone (EPH116) exhibited above-average inhibition of colon carcinoma (IC(50) = 1.3-4.56 nM); EPH52 and EPH116 also exhibited above-average inhibition of melanoma cells. As shown with human liver microsomes, EPH116 is only moderately metabolized. The compound inhibited the growth of human colon cancer xenografts in nude mice in a dose-dependent manner. Thiosemicarbazones derived from 2-formylpyridines have been shown to be inhibitors of ribonucleotide reductase (RR). The following results show that RR is not the target of the novel compounds: cells overexpressing the M2 subunit of RR and resistant to the RR inhibitor hydroxyurea are not cross-resistant to the novel compounds; inhibition of RR occurs at 6- to 73-fold higher drug concentrations than that of inhibition of cell proliferation; the pattern of cell cycle arrest in S phase induced by the RR inhibitor hydroxyurea is not observed after treatment with the novel compounds; and a COMPARE analysis with the related compounds 2-acetylpyrazine benzothiazol-2-ylhydrazone (EPH95) and 3-acetylisoquinoline benzoxazol-2-ylhydrazone (EPH136) showed that the pattern of these compounds is not related to any of the standard antitumor drugs. Therefore, these novel compounds show inhibition of colon cancers and exhibit a novel mechanism of action.

Synthesis, cytotoxicity, and antitumor activity of copper(II) and iron(II) complexes of (4)N-azabicyclo[3.2.2]nonane thiosemicarbazones derived from acyl diazines.

A series of thiosemicarbazones (TSCs) (bearing a (4)N-azabicyclo[3.2.2]nonane moiety) derived from 3-acylpyridazines, 4-acetylpyrimidines, and 2-acetylpyrazines (1-8) were synthesized as potential antitumor agents. TSCs 1-8 exhibited potent cytotoxic activity against human acute lymphoblastic leukemia CCRF-CEM cells (IC(50) = 0.05-0.77 microM) and colon adenocarcinoma HT-29 cells (IC(50) = 0.011-2.22 microM). Copper II complexes of TSCs 1-8 showed significant improvement in cytotoxic activity against HT-29 cells (IC(50) = 0.004-1.51 microM) by a factor of 3. However, complexation of ligands 1, 2, 4, and 6 with Fe(II) results in lowering of cytotoxic activity by a factor of approximately 7. In clonogenic assays involving human tumor cells of different tumor origins, compounds 5, 7, 8, and their copper complexes 5Cu(II), 7Cu(II), and 8Cu(II) exhibited remarkable cytotoxic activities with mean IC(50) values of 6, 0.18, 1, 1, 0.37, and 0.37 nM, respectively. In particular, the compounds were highly effective against human colon carcinoma and large and small cell lung carcinoma cells. The TSC derivative 5 was evaluated in vivo in nude mice bearing LXFL 529 human large cell lung carcinoma cells. With respect to antitumor activity, application of 30 mg/kg/d resulted in moderate inhibition (42%) of tumor growth. No effect on tumor growth was observed at a dose of 10 mg/kg/d. However, a dose of 40 or 60 mg/kg/d resulted in 50 and 75% death, respectively, in the treated mice, indicating the high toxicity of these compounds. Using human liver microsomes, compound 5 was found to be rapidly and highly metabolized in vitro. In actual fact, only 2% of the unmetabolized compound could be detected in the incubation medium after 5 min. The IC(50) for cell proliferation (0.006-0.022 microM) elicited by these compounds is much lower than that of the inhibition of [(14)C]cytidine incorporation into DNA (0.18-3.32 microM). These compounds are also noncell cycle specific agents. Interestingly, compounds 5, 5Cu(II), and 8 were found to be potent inducers of apoptosis in Burkitt's lymphoma cells.

Pyridazines 91. Synthesis of substituted tri- and tetracyclic compounds bearing a pyridazine core and their biological evaluation as antimycobacterial agents.

Starting from substituted 3,6-dichloropyridazine-4-carboxamides (2, 3) tri- and tetracyclic compounds (4, 5) could be smoothly prepared. Structural modifications of interest with regard to biological activity were performed by N-alkylation and reductive dehalogenation. The new substituted heterocyclic compounds were screened as antimycobacterial agents; the influence of the substitution pattern on activity is discussed.

Investigations on the mechanism of action of the novel antitumor agents 2-benzothiazolyl, 2-benzoxazolyl, and 2-benzimidazolyl hydrazones derived from 2-acetylpyridine.

2-Acetylpyridine hydrazone derivatives of benzothiazole, benzoxazole, and benzimidazole were found to exhibit potent cytotoxic activity against the growth of suspended leukemia and lymphomas. They were also active in a number of solid tumor screens, e.g. HeLa uterine carcinoma, SOS bone osteosarcoma, lung MB9812, lung A549, Mcf-7 breast growth. In L1210 lymphoid leukemia cells the compounds preferentially inhibited RNA synthesis followed by DNA synthesis at 100 microM after 60 min. The reduction of de novo purine synthesis by the compounds at the regulatory sites PRPP-amido transferase, IMP dehydrogenase and dihydrofolate reductase was responsible for the suppression of nucleic synthesis. Other minor sites where the agents have metabolic effects were thymidylate synthetase and thymidine kinase which would be additive with the overall inhibition of cell growth. The ct-DNA studies suggest that the compounds also interacted with the DNA molecule itself, probably affecting template activity.

Synthesis of N-aryl-N'-heteroaryl-substituted urea and thiourea derivatives and evaluation of their anticonvulsant activity.

Starting from amino(di)azines and 2-chloro-6-methyliso(thio)cyanate a series of aryl-substituted urea and thiourea derivatives was prepared and screened as potential antiepileptics. Among the new derivatives tested, only 2b and 3c exhibited adequate anticonvulsant effects, whereas 3d and 4d were found to be convulsants per se.

Pyridazines 82. Synthesis of pyridazino [3,4-b][1,5]benzodiazepin-5-ones and their biological evaluation as non-nucleoside HIV reverse transcriptase inhibitors.

Starting from 3,6-dichloro-N-(2-chloro-5-nitrophenyl)-pyridazine-4-carboxamide (7) a series of 6,11-dialkylated pyridazino- [3,4-b][1,5]benzodiazepin-5-ones with a 3-chloro-8-nitro, 8-amino, 8-acetylamino, or 8-chloro substitution pattern was prepared via N-alkyl-3-alkylamino-6-chloro-N-(2-chloro-5-nitrophenyl) -pyridazine-4-carboxamides. The new compounds were screened as non-nucleoside reverse transcriptase inhibitors. The influence of the substitution pattern in compounds 10-13 on inhibitory potency is discussed.

Azinyl and diazinyl hydrazones derived from aryl N-heteroaryl ketones: synthesis and antiproliferative activity.

A series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl methanones was prepared in search for potential novel antitumor agents. The stereochemistry of these compounds was established by means of NMR spectroscopy. Antiproliferative activity was determined in a panel of human tumor cell lines (CCRF-CEM, Burkitt's lymphoma, HeLa, ZR-75-1, HT-29, and MEXF 276L) in vitro. Generally, the new compounds were found to be more potent (IC50 = 0.011-0.436 microM) than the ribonucleotide reductase inhibitor hydroxyurea (IC50 = 140 microM). Most of the compounds exhibited the highest activity against Burkitt's lymphoma with an IC50 of 0.011-0.035 microM. [14C]Cytidine incorporation into DNA was quantitated for selected hydrazones (Z-A, E-1, Z-3, Z-4, E-5, Z-5, E-13, E-18, Z-19, Z-24, and E-26) as a measure of the inhibition of ribonucleotide reductase in Burkitt's lymphoma cells. The E-configurated compounds were found to inhibit [14C]cytidine incorporation to a greater extent (IC50 = 0.67-5.05 microM) than the Z-isomers (IC50 = 7.20 to > 10 microM). Principal component analysis of the IC50 values obtained for inhibition of cell proliferation revealed that the cell lines tested can be grouped into three main families showing different sensitivities toward the compounds in our series [(i) CCRF-CEM, Burkitt's lymphoma, and Hela; (ii) HT-29; and (iii) MEXF 276 L].

On the bioisosteric potential of diazines: diazine analogues of the combined thromboxane A2 receptor antagonist and synthetase inhibitor Ridogrel.

In this SAR study the bioisosteric potential of diazines in the field of combined antithrombotic thromboxane A2 synthetase inhibitors and receptor antagonists was investigated. In this context, two series of (E)- and (Z)-omega-[[(aryldiazinylmethylene)amino]oxy]alkanoic acids were synthesized of which pentanoic acid derivatives with a 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl group were found to exhibit this dual activity, while 4-pyrimidinyl as well as 3-pyridazinyl analogues showed only receptor antagonistic activity and 2-pyrimidinyl congeners were inactive. In the series of diazine analogues of Ridogrel (1), replacement of the 3-pyridyl group by a 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl moiety led to compounds that inhibit thromboxane A2 synthetase in gel-filtered human platelets comparable to 1 (IC50 of 0.006, 0.016, and 0.039 microM, respectively, versus 0.007 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that these diazine analogues block the thromboxane A2 receptor with an IC50 of 11, 6.0, and 1.5 microM, respectively. This compares well with the IC50 = 1.7 microM of 1. Finally, testing of inhibition of collagen-induced platelet aggregation in human platelet aggregation in human platelet-rich plasma with 2-pyrazinyl, 4-pyridazinyl, or 5-pyrimidinyl congeners of Ridogrel indicated that these heteroaromatic moieties may serve as bioisosteric substitutes of a 3-pyridyl group in dual-acting antiplatelet agents.

On the synthesis of pyridazomycin congeners. Part 2: L-alpha-Amino acids bearing a terminal azinium or diazinium system.

The synthesis of a variety of novel compounds structurally related to the antimicrobial natural product pyridazomycin via alkylation of appropriate azine and diazine derivatives is reported. Based on the results of preliminary antimicrobial tests the dependence of antimicrobial activity from several structural features of pyridazomycin is discussed.

Pyridazines, LXXII: On the synthesis of azinium and diazinium compounds structurally related to pyridazomycin.

Preparation of series of pyridine-, pyridazine-, and pyrazine-derived carboxamides bearing at the ring N-atom an alkyl side-chain with a terminal carboxylic group (7-11) or with a terminal acetylamino malonic ester moiety (13-17, 19-23) is described. Two desaza-pyridazomycin derivatives (24, 26) and homologs thereof (25, 27) were synthesised. The novel compounds which are structurally related to the antifungal antibiotic pyridazomycin were screened for antifungal activity: preliminary in vitro tests showed no activity.

[AgNOR expression in skin tumors. Studies of melanocytic, epidermal and fibrohistiocytic lesions]. / AgNOR-Expression bei Hauttumoren. Untersuchungen an melanozytären, epidermalen und fibrohistiozytären Läsionen.

Nucleolus organizer regions are segments of DNA-coding ribosomal genes, which can be visualized histologically by a special silver staining technique as so-called AgNORs. Determination of the number and size of AgNORs provides quite a precise reflection of current cellular proliferation activity. We have examined the AgNOR expression in 228 melanocytic, epidermal and fibrohistiocytic lesions. Dysplastic naevi and Spitz naevi showed a significantly lower AgNOR expression than melanomas, and there were analogous differences between keratoacanthomas and carcinomas. Furthermore, there were also significant differences between the groups of melanomas and carcinomas, as there were between benign and non-benign fibrohistiocytic lesions. The AgNOR expression in more cellular fibrohistiocytic lesions differed from at in more fibrous lesions. Analysis of AgNOR areas was not so informative as AgNOR counting. The results of PCNA examinations correlated with the degree of AgNOR expression. It was found that the AgNOR technique is useful in differential diagnosis and for histological determinations of proliferation activity in melanocytic, epidermal and fibrohistiocytic lesions. The AgNOR method is easy to apply and can be applied in paraffin-embedded tissue.

Determination of epidermal proliferative activity in experimental mouse tail test by AgNOR analysis.

Nucleolar organizer regions are segments of DNA coding ribosomal genes, which can be histologically detected by silver technique as so-called AgNORs. The estimation of AgNOR number and AgNOR size are currently under investigation as markers of cellular proliferation activity. We therefore examined the epidermal AgNOR expression after topical application of different antiproliferative compounds using the mouse tail test. The epidermis of 0.025% fluocinolone acetonide-treated mouse tails had the lowest AgNOR expression. Pretreatment with 0.2% Anthralin, 1% propyl gallate and 2% 3,4-hexaalkylbenzoylacrylic acid, an experimental phospholipase-A2 inhibitor, also revealed significant inhibition effects of epidermal AgNOR expression. Native and petrolatum-treated epidermis as control showed the highest AgNOR expression. The AgNOR results in basal cells proved to be more informative than these in the stratum spinosum, the best parameter was the AgNOR number. The obtained results were closely related to the values of the corresponding studies of PCNA expression. The AgNOR method seems to be useful for estimation of antiproliferative efficiency of pharmacological substances. This technique is simple in handling and can be applied using paraffin-embedded tissue sections.

Pyridazine analogs of piperidinylbenzophenone immunomodulators. 70th communication: syntheses and reactions of pyridazines.

Starting from conveniently accessible halogen-substituted phenyl pyridazinyl ketones, a series of piperidinyl-substituted diazabenzophenones was prepared. The new compounds were screened for immunomodulatory activity.

[Histological grading of ductal pancreatic carcinomas. Relationship to silver staining of NOR-associated proteins and to p53 immunoreactivity]. / Histologisches Grading duktaler Pankreaskarzinome. Beziehung zur Silberfärbung NOR-assoziierter Proteine und zur p53-Immunoreaktivität.

The histological grading of 29 ductal pancreatic carcinomas was compared with that of silver stained NOR-associated proteins and with p53 immunoreactivity. The AgNOR number (NZ) and AgNOR areas (NF) of 10 grade I carcinomas, 13 grade II carcinomas and 6 grade III carcinomas were investigated by image analysis. Furthermore, the protein expression of the p53 gene was examined by immunohistochemistry. A significant difference was found in both AgNOR parameters between grade I, grade II and grade III carcinomas (NZ: p < 0.01; NF: p < 0.05; grade I carcinomas: NZ 5.1/NF 8.2 microns 2; grade II carcinomas: NZ 6.0/NF 11.3 microns 2 grade III carcinomas: NZ 8.4/NF 15.2 microns 2). By contrast no relation was found between histological grading and p53 immunoreactivity. In 56% of all carcinomas positive evidence was found of the p53 gene product. (6 grade I carcinomas, 7 grade II carcinomas, 3 grade III carcinomas). 44% of the cases were p53-negative (4 grade I carcinomas, 6 grade II carcinomas, 3 grade III carcinomas). This study demonstrates that the AgNOR technique is useful in the grading of ductal pancreatic carcinomas, whereas the expression of the p53 gene product is not.

[Use of the AgNOR method for the differential diagnosis of tumors and for the quantification of non-neoplastic epidermal hyperproliferation in dermatohistology]. / Einsatz der AgNOR-Methode für die Tumor-Differentialdiagnostik und für die Quantifizierung nicht-neoplastischer epidermaler Hyperproliferationen in der Dermatohistologie.

The AgNOR number (NN) and the AgNOR quotient (NQ) of 228 melanocytic, epidermal and fibrohistiocytic lesions were investigated by image analysis. In melanocytic nevi, dysplastic nevi and Spitz nevi, significantly less AgNORs were seen than in malignant melanomas and melanomas in situ. Significant differences in AgNOR expression were detected between the subtypes of melanoma, too. Epidermal carcinomas showed a significantly higher AgNOR expression than keratoacanthomas and other benign tumors. Between the G1 carcinomas and the G2 and G3 groups, an increase of the NN and NQ was found. Actinic keratosis demonstrated a significantly lower AgNOR expression than carcinomas and--like M. Bowen--higher NN and NQ than the benign lesions. The lowest AgNOR results of fibrohistiocytic lesions were registered in regressive palmar fibromatosis and in scars, the highest in malignant fibrous histiocytomas. Between dermato-fibrosarcoma protuberans, atypical fibroxanthoma and malignant fibrous histiocytomas, significant differences were found to exist. Furthermore we examined the suitability of the AgNOR technique for quantification of epidermal cell kinetics in non-neoplastic epidermal hyperproliferations. Especially in the grading of psoriasis this technique is helpful. First convincing results could be achieved for the use of the AgNOR method in the verification of antiproliferative effects of new substances in an experimental mouse tail test. This study demonstrated that the AgNOR technique in addition to the dermatohistological differential diagnosis of tumors is useful for the quantification of antiproliferative effects of different treatments in hyperproliferative epidermal dermatosis.

Analytic morphology in clinical and experimental dermatology.

During the past several years, quantitative morphology has gained increasing attention in diagnostic pathology and in certain research applications. In the field of dermatopathology, quantitative morphology has been applied to numerous problems, ranging from the interactive measurement of nuclear contours to fully automated, high-resolution image analysis of ultrastructural micrographs. Dermatologic applications are reviewed, and potential developments in the future are briefly outlined.