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The present study describes an in vivo bioavailability experiment for genistein and its glycoside genistin, either as pure compounds or from a soy protein isolate extract, using freely moving unanesthetized rats with a cannulation in the portal vein. The results show that genistein is readily bioavailable, being observed in portal vein plasma at the first point of detection at 15 min after dosing. The AUC(0-24h) values for total genistein and its conjugates were 54, 24, and 13 microM h for genistein, genistin, and an enriched protein soy extract, respectively. These results indicate that the bioavailability of genistein is higher for the aglycon than for its glycoside. Genistin is partly absorbed in its glycosidic form. It is concluded that bioavailability studies based on portal vein plasma levels contribute to insight into the role of the intestine and liver in deglycosylation and uptake characteristics of glycosylated flavonoids.

The commercialisation of GM crops in Europe is practically non-existent at the present time. The European Commission has instigated changes to the regulatory process to address the concerns of consumers and member states and to pave the way for removing the current moratorium. With regard to the safety of GM crops and products, the current risk assessment process pays particular attention to potential adverse effects on human and animal health and the environment. This document deals with the concept of unintended effects in GM crops and products, i.e. effects that go beyond that of the original modification and that might impact primarily on health. The document first deals with the potential for unintended effects caused by the processes of transgene insertion (DNA rearrangements) and makes comparisons with genetic recombination events and DNA rearrangements in traditional breeding. The document then focuses on the potential value of evolving "profiling" or "omics" technologies as non-targeted, unbiased approaches, to detect unintended effects. These technologies include metabolomics (parallel analysis of a range of primary and secondary metabolites), proteomics (analysis of polypeptide complement) and transcriptomics (parallel analysis of gene expression). The technologies are described, together with their current limitations. Importantly, the significance of unintended effects on consumer health are discussed and conclusions and recommendations presented on the various approaches outlined., (Copryright 2004 Elsevier Ltd.)

Genistein receives much attention because of its potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of this compound like, for instance, their intestinal uptake by humans and systematic bioavailability. In this study, the fate of the absorption of genistein and its glycoside has been analysed in various isolated perfused gut segments of the rat. In all perfused gut segments the transport of genistein was higher compared to its glycoside. Furthermore, it appeared that the resorbate (i.e. serosal side) concentration of genistein was the highest in ileac segments, whereas the transport of genistein in the various other segments tested showed no difference between intestinal compartments. Less than 0.2% of genistin appeared in the resorbate fluid of all isolated gut segments. The main site of metabolism of genistein and its glycoside appears to be located in the jejunal compartment of the rat gut. About 38% of genistein and about 29% of genistin metabolised within 2h of perfusion. In the ileac and colonic intestinal segments, genistein metabolised for only 10%. For the first time, this study demonstrated that genistin could be metabolised by epithelial cells present in isolated colonic segments. However, the metabolites of genistin did not occur at the serosal side (the resorbate) of isolated colonic segments. We assume that there is no absorption of genistin and/or its metabolites in or through colonic tissue of the rat.

Genistein and daidzein receive much attention because of their potential to prevent hormone-related cancer and cardiovascular diseases. Limited information is available on the pharmacokinetics of these compounds like, for instance, intestinal uptake by humans and systematic bioavailability. In this study the transport and metabolism of genistein, daidzein and their glycosides has been compared in various cellular models for intestinal absorption such as human colonic Caco-2, rat small intestinal IEC-18 and human immortalized colon HCEC cell lines. Genistein and daidzein were taken up by Caco-2, IEC-18 and HCEC cells and transported to almost same rate and extents. Glycosides were transported across IEC-18 and HCEC monolayers, but not across Caco-2 cells. In Caco-2 and IEC-18 cells, the glycosides were metabolized to their respective aglycones. Furthermore, it was shown that genistein and daidzein were glucuronidated and sulfated in Caco-2 cells, to glucuronidated forms in IEC-18 cells and to sulfated conjugates in HCEC cells. The results of this study compared with reported in vivo data indicate that Caco-2 cells are a valuable model for studying intestinal transport and metabolism of isoflavones.

Safety assessment of genetically modified food crops is based on the concept of substantial equivalence, developed by OECD and further elaborated by FAO/WHO. The concept embraces a comparative approach to identify possible differences between the genetically modified food and its traditional comparator, which is considered to be safe. The concept is not a safety assessment in itself, it identifies hazards but does not assess them. The outcome of the comparative exercise will further guide the safety assessment, which may include (immuno)toxicological and biochemical testing. Application of the concept of substantial equivalence may encounter practical difficulties: (i) the availability of near-isogenic parental lines to compare the genetically modified food with; (ii) limited availability of methods for the detection of (un)intended effects resulting from the genetic modification; and (iii) limited information on natural variations in levels of relevant crop constituents. In order to further improve the methodology for identification of unintended effects, new 'profiling' methods are recommended. Such methods will allow for the screening of potential changes in the modified host organism at different integration levels, i.e. at the genome level, during gene expression and protein translation, and at the level of cellular metabolism.

Previous studies suggest that vitamin A deficiency may induce or intensify inflammatory changes in the rat gastrointestinal system. The present study was designed to compare the expression profiles of rat models of vitamin A deficiency and induced colitis. cDNA-microarray technology was used to determine the genes involved in the inflammatory processes in the two models. mRNA was extracted from colons of rats that were vitamin A deficient, vitamin A supplemented (control), or had 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, reverse-transcribed into fluorescence-labeled cDNA and hybridized onto microarrays containing a duplicate set of 1152 cDNAs, derived mainly from the colon carcinoma cell line Caco-2. Differential gene expression was detected in vitamin A deficiency and in TNBS-induced colitis vs. control. beta-Actin, translation initiation factor A4 and translation elongation factor 1, ornithine decarboxylase (ODC) and keratin 19 were markedly down-regulated, whereas spermidine/spermine N1-acetyltransferase (SSAT) and polyubiquitin (UbC) were up-regulated in both vitamin A-deficient rats and those with TNBS-induced colitis. The strong association between the differential gene expression in the two animal models, compared with the control, suggests that deficiency of vitamin A causes inflammatory changes in the rat colon that are similar to processes occurring in colitis. Further investigation is required to elucidate the importance of each of the regulated genes to the pathology of colitis and vitamin A inadequacy.

International consensus has been reached on the principles regarding evaluation of the food safety of genetically modified plants. The concept of substantial equivalence has been developed as part of a safety evaluation framework, based on the idea that existing foods can serve as a basis for comparing the properties of genetically modified foods with the appropriate counterpart. Application of the concept is not a safety assessment per se, but helps to identify similarities and differences between the existing food and the new product, which are then subject to further toxicological investigation. Substantial equivalence is a starting point in the safety evaluation, rather than an endpoint of the assessment. Consensus on practical application of the principle should be further elaborated. Experiences with the safety testing of newly inserted proteins and of whole genetically modified foods are reviewed, and limitations of current test methodologies are discussed. The development and validation of new profiling methods such as DNA microarray technology, proteomics, and metabolomics for the identification and characterization of unintended effects, which may occur as a result of the genetic modification, is recommended. The assessment of the allergenicity of newly inserted proteins and of marker genes is discussed. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from genetically modified crops. It is concluded, among others that, that application of the principle of substantial equivalence has proven adequate, and that no alternative adequate safety assessment strategies are available.

The isoflavonoid genistein inhibits mitosis and increases apoptosis in a variety of tumour cell lines in vitro, and may exert anticarcinogenic effects in vivo. To assess its effects on the colon, rats were fed a semi-synthetic control diet, or similar diets enriched with genistein (0.25 g/kg), either as the pure isoflavone or as part of a soya protein isolate, for 7 days before receiving subcutaneous injections of saline or 1,2-dimethylhydrazine (DMH). After 48 h, rats given saline were killed and samples of their small and large intestinal mucosa were obtained for assessment of crypt cell mitosis and apoptosis by visual analysis of isolated intact crypts. Rats given DMH were fed control diet and killed after 48 h for assessment of crypt cytokinetics or maintained for 42 days then killed and their colonic mucosa analysed for aberrant crypt foci (ACF). Two further groups were given control diet before DMH, followed by the genistein or soya-based diet for 42 days before assessment of ACF. Neither genistein nor soya protein isolate had a significant effect on crypt cell mitosis or apoptosis in untreated rats, or on the proliferative response to treatment with DMH. However, consumption of pure genistein or the soya protein isolate before treatment with DMH was associated with a 3-fold (P < 0.001) or 2-fold (P < 0.05) increase, respectively, in ACF in the distal colon. There was no significant effect of genistein or soya protein isolate given after DMH treatment. We conclude that genistein has no detectable effect on colonic crypt mitosis or apoptosis in the rat in vivo, but that it promotes induction of ACF by an as yet undefined mechanism when fed immediately before treatment with DMH.

A common element in designed guidelines for assessment of the food safety of transgenic crops is centred on a comparative analytical analysis with conventionally bred crop plants, assuming that these products have a long history of safe use (i.e. OECD-principle of substantial equivalence). In this study we examine the utility of an off-line combination of 400 MHz proton (1H)-NMR spectroscopy and liquid chromatography (LC) for the multi-component comparison of low-molecular weight compounds (i.e. chemical fingerprinting) in complex plant matrices. The developed NMR-methodology can contribute to the demonstration of substantial equivalence by its ability to compare possible compositional alterations in a novel food crop with respect to related non-transgenic reference lines. In this respect a hierarchical approach is proposed by comparing the chemical fingerprints of the transgenic crop plant to those of: (1) isogenic parental or closely related lines bred at identical and multiple sites; (2) extended ranges of commercial varieties of that plant; and (3) downstream processing effects. This is of importance to assess the likelihood that some of the statistical differences in a transgenic crop plant may be false positives due to chance alone or arose from natural genetic and/or physiologic variations.

In this study information was obtained on bioavailability of genistein, daidzein and their glycosides in human intestinal epithelial Caco-2 cells grown on semi-permeable filters. The integrity of Caco-2 monolayers was confirmed by transepithelial electrical resistance measurements and by determination of the permeability of the radioactive marker polyethylene glycol (PEG4000). After 6 h approximately 30-40% of genistein and daidzein added at the apical side was transported to the basolateral side and this level was maintained for 24 h, The glycosides were barely transported through the Caco-2 cells. No significant metabolism of genistein and daidzein in the Caco-2 cells occurred, whereas the glycosides were mainly metabolised to their respective aglycones. Obviously, our data indicates that Caco-2 cells contain an endogenous glycosidase activity.

The intestinal transport and metabolism of quercetin and various sugar-conjugates were quantified in in vitro and in vivo model systems. The nature of the sugar moiety at the C3 and C4' position had no significant effect on the rate of transport. At the 10 microM level, quercetin and glycosides with sugars at position 3 were determined to be glucose transport carrier inhibitors.

A selective and specific high-pressure liquid chromatographic (HPLC) method for the simultaneous assay of Clanfenur and its metabolites in biological fluids of interest has been developed which is suitable for routine analysis, using micro volumes (0.1 ml) of plasma samples only. After protein precipitation the extract is analysed by reversed-phase HPLC with UV detection. Excellent recovery, linearity, accuracy and precision (less than 5% for plasma) are achieved by the assay which is able to quantify Clanfenur and its metabolites in plasma at concentrations between 0.025 and 5.0 mg l-1.

Albumin-heparin microspheres were prepared by a two-step process which involved the preparation of a soluble albumin-heparin conjugate, followed by formation of microspheres from this conjugate or by a double cross-linking technique involving both coupling of soluble albumin and heparin and microsphere stabilization in one step. The first technique was superior since it allowed better control over the composition and the homogeneity of the microspheres. Microspheres could be prepared with a diameter of 5-35 microns. The size could be controlled by adjusting the emulsification conditions. The degree of swelling of the microspheres was sensitive to external stimuli, and increased with increasing pH and decreasing ionic strength of the medium.

The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M(r) range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [125I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (Ka of 4.7 +/- 0.2 x 10(9) M-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M(r) 24,000. The functional status of PRL- and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.

A chemical analysis was instigated to investigate the identity of the luteinizing hormone (LH)-like immunoactivity present in ovine pineal protein homogenates. Isolation of pineal LH-like material was accomplished using a 0.1 M ammonium sulphate (pH 4.0) extraction followed by anion-exchange chromatography. The resulting 3.0 M ammonium sulphate precipitate containing 70% of the LH-like immunoactivity was refractionated by cation-exchange and Sephadex G-100 chromatography. Analysis of the pattern of recovered LH-like immunoactivity in the Sephadex G-100 eluate indicated the presence of molecular weight (MW) less than 60,000 besides MW 21,000 species of LH-like proteins. Bioactivity was tested in the rat Leydig cell steroidogenesis assay. In terms of steroid production, the activity was associated with the MW 21,000 LH-like proteins only. Further purification by CM-Sephadex chromatography and gel permeation HPLC was conducted in order to determine whether the physicochemical properties of pineal LH-like material represented endogenous LH, synthesized and released by the ovine pituitary. It is concluded by a variety of means, including polyacrylamide gel electrophoresis, and amino acid and carbohydrate analyses, that at least two molecular forms of immunoactive LH-like proteins occur in ovine pineal tissue. The MW 21,000 forms showed much similarity with ovine adenohypophyseal LH or with a complex mixture of its subunits. These observations contribute to the understanding of endocrine-endocrine transducing events that may occur in this organ.

We have studied the cellular pharmacokinetics of carboplatin (CBDCA), as part of the evaluation of the antitumor activity of CBDCA in cancers limited to the peritoneal cavity in comparison with cisplatin (cDDP). The uptake of CBDCA into L1210 (lymphosarcoma), CC531 (colonic carcinoma), COV413.B (human ovarian carcinoma) and NB1 (human neuroblastoma) cells was 1.5 to 13 times lower than the uptake of cDDP. The uptake of CBDCA into human ovarian carcinoma cells, taken directly from patients, was also 8-20 times lower than cDDP. Platinum concentrations, expressed as a percentage of the total intracellular Pt concentration, were similar for CBDCA and cDDP in cytosol and nucleus/membrane fractions. A second major difference between the drugs was their binding to DNA. Less CBDCA-DNA than cDDP-DNA adducts were formed after incubation at equimolar amounts of drug with isolated salmon sperm DNA (5-25 times less). A 16-69 times higher concentration of CBDCA than cDDP was needed to induce similar changes in cell growth activity (50% [3H]thymidine inhibition) in CC531 and COV413.B cells, indicating that equitoxicity can only be achieved when tumor cells are exposed to higher concentrations of CBDCA than cDDP. Similar toxicity was achieved in CC531 cells after incubation with a 16-fold higher CBDCA dose than cDDP. Comparable intracellular platinum concentrations, however, were obtained with a 10-fold higher CBDCA dose, suggesting that cellular pharmacokinetics of the drugs are different. Regarding drug uptake and pharmacokinetics the mechanism of action of CBDCA differed from cDDP at a cellular level.

A number of polyclonal antibodies specific for DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) were obtained from the sera of New Zealand white rabbits immunized with BPDE-DNA, complexed with methylated bovine serum albumin (mBSA). Monoclonal antibodies were developed by fusion of mouse myeloma cells with spleen cells isolated from BALB/c mice immunized with the same complex of BPDE-DNA and mBSA. These antibodies have been characterized for specificity in a highly sensitive, enzyme-linked immunosorbent assay (ELISA). All antibodies showed a very high affinity for single-stranded BPDE-DNA, but had lower affinity towards native BPDE-DNA. The affinity for the free mononucleoside BPDE-dG was at least 100-fold lower than that for BPDE-DNA, and no affinity was detected for BP tetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene. A high cross reactivity was observed with DNA modified with (+/-)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene++ +. Using five different antibodies, monoclonal or polyclonal, we observed that the antibody affinity for BPDE-DNA was dependent on the level of modification; in the competitive ELISA as little as 4 fmol BPDE-DNA (50 pmol/micrograms) was sufficient for 50% inhibition with our best antisera, but 17 fmol of the adduct was required when [3H]BPDE-DNA of low modification (1-10 fmol/micrograms) was used as inhibitor. When samples of [3H]BP-DNA isolated from the livers of mice, treated i.p. with different doses of [3H]BP were examined by competitive ELISA and calibrated with [3H]BPDE-DNA of low modification (1-10 fmol/micrograms), binding values calculated from the immunoassay were in good agreement with those obtained from radioactivity measurements. In contrast, when this DNA was quantitated in competitive ELISA using highly modified BPDE-DNA as standards, values by ELISA were 20-40% of those obtained by radioactivity. These results indicate that the use of serially diluted BPDE-DNA of high modification as standard competitor in the ELISA will lead to erroneous results in the measurement of adducts in DNAs modified to a low extent (biological samples). The property of antisera specific for BP-DNA, recognizing highly modified DNA more efficiently than DNA modified to a low extent, may be common to all antisera elicited against highly modified DNA immunogens. Therefore we conclude that antibody affinity must be tested also with DNA samples of low modification, obtained either in vitro or in vivo.

The nonapeptide delta-sleep-inducing peptide (DSIP) has been isolated from venous blood of rabbits induced to sleep. Numerous reports have described sleep as well as extra-sleep effects. Radiochemical and immunochemical data suggest a relationship of DSIP with the pineal gland supported by interactions of this peptide with pineal functions such as the serotonin N-acetyltransferase activity. In order to demonstrate the natural occurrence of DSIP-like material associated with high Mr proteins in the ovine pineal, organs were water-extracted and fractionated by ultrafiltration and gel filtration. Radioimmunoassay (RIA) for DSIP-like fragments of the fractions revealed considerable amounts of pineal DSIP-like immunoreactivity (DSIP-LI) apparently existing in small as well as large molecular forms. Acidification of large DSIP-LI forms resulted in the elution from Sephadex G-50 of Mr less than or equal to 1,000 DSIP-like material. This free DSIP-LI form coeluted with the synthetic DSIP nonapeptide from microBondapak C18 on high-performance liquid chromatography. The results, therefore, appear to indicate the presence of a (biospecific) noncovalent intermolecular interaction of DSIP (1-9) with proteins (Mr greater than or equal to 10,000) of the ovine pineal gland.

The milk-ejecting response of lactating mouse mammary gland tissue to ovine pineal extracts indicated the presence of a neurohormone-like bioactivity in this tissue. After successive fractionation on gel permeation chromatography and reversed-phase liquid chromatography (HPLC) in conjunction with radioimmunoassays (RIA), it was demonstrated that the milk-ejection response to ovine pineal components with an Mr less than 1,000 corresponded to a biologically active peptide sequence that probably differs from that of arginine vasopressin, arginine vasotocin, and oxytocin and from peptides with a COOH-terminal Pro-Arg-Gly-amide ending. Gel permeation chromatography in formic acid appeared also to indicate the presence of a noncovalent interaction of the neurohormone-like bioactivity with proteins (Mr greater than 25,000) of the pineal.

1. The acyl-CoA:lysophosphatidylcholine acyltransferase system in rat lung microsomes was found to utilize selectively 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine when compared with 1-[9,10-3H2]stearoyl-sn-glycero-3-phosphocholine. This result was found with either palmitoyl-CoA, linoleoyl-CoA or an equimolar mixture of these acyl donors and confirms recent data reported by Holub, Piekarski and Possmayer (Can. J. Biochem. 58 (1980) 434-439). 2. The selective utilization of palmitoyl lysophosphatidylcholine from a mixture of lysophosphatidylcholine species may cause an increased isotopic ratio in phosphatidylcholine when compared with that of total lysophosphatidylcholine. Thus, when rats were injected with a single doubly labelled species, i.e. 1-[9,10-3H2]palmitoyl-sn-glycero-3-phospho[methyl-14C]choline, the isotopic ratio in both total and disaturated phosphatidylcholine from lung was nearly identical to that of the injected substrate. This suggested a direct acylation by lung acyl-CoA:lysophosphatidylcholine acyltransferases. By contrast, when a mixture of 1-[9,10-3H2]palmitoyl-sn-glycero-3-phospho[methyl-14C]choline and 1-stearoyl-sn-glycero-3-phospho[methyl-14C]choline was injected, the 3H/14C ratio in disaturated lung phosphatidylcholine increased to about 1.4-fold that of the injected substrate. 3. These data indicate that increased isotopic ratios in disaturated phosphatidylcholine of lung tissue, after intravenous injection of lysophosphatidylcholine, do not necessarily point to the involvement of lysophosphatidylcholine:lysophosphatidylcholine transacylase in disaturated phosphatidylcholine formation.

In the rat pineal gland the mechanism of release of secretory material was studied ultrastructurally after incubating tissues in Ringer solution containing tannic acid. The results indicate that pinealocytes release the contents of secretory vesicles into the extracellular space via exocytosis, a phenomenon that has not been visualized previously in this cell type. This finding may reflect release of polypeptides by the pineal gland.

Former work has shown that crude extracts of ovine pineal glands probably exert a stimulating activity on the release of gonadotropins of anterior pituitaries in vitro. By aqueous extraction followed by ultrafiltration through anisotropic membranes high Mr (above 100,000 daltons) fractions were obtained, which exhibit a stimulating effect on the levels of gonadotropins in the medium of either cultured pituitary cells or anterior hemipituitaries in short-term culture. Partial purification of a pineal luteinizing hormone release stimulating factor was accomplished by Sephadex G-150 filtration with a biopotency of 226 +/- 23 micrograms LH-RP-1 equivalents per mg protein and without an affinity for binding to anti-LHRH or anti-LH antibodies. The present data substantiate that high Mr forms, slightly heavier than authentic pituitary LH (Mr 23,000 daltons) and therefore not identical to the hypothalamic decapeptide LH-RH, represent ovine pineal factors which can increase the concentration of LH in the medium of cultured anterior pituitaries, but does not influence the secretion of prolactin in vitro.

Granular vesicles are present in pinealocytes and in rudimentary photoreceptor cells of many vertebrates, sometimes in large amounts. Their dense cores have been shown to store proteinaceous compounds, but the way they are released remains speculative. The aim of this study was to demonstrate whether or not exocytosis is the mechanism by which secretory products stored within granular vesicles are released. Therefore, a method has been used allowing a clear ultrastructural study of secretory products by exocytosis, even in tissues in which this process of secretion is quite rare and/or very slow. Exocytotic figures have been clearly demonstrated in the three species studied: golden hamster, snake, and parakeet. Nevertheless, they were never commonly observed as it was the case in neurohypophysis, even in such animals as the parakeet and snake, in which granular vesicles are very numerous. The possible reasons of this observation are discussed.

Using an aqueous extraction followed by ultrafiltration through Amicon Diaflo membranes, two ovine pineal fractions were obtained, which contain immunoreactive neurophysin. The presence of neurophysin was monitored by radioimmunoassay, employing an antiserum raised against pituitary bovine neurophysin and selected because it reacts with neurophysins of many other mammals. From 50 g of wet ovine pineal glands 552 micrograms of immunoreactive neurophysins were obtained. About 5% of these immunoreactive neurophysins are eluted from three different Sephadex columns with an elution volume corresponding to Mr above 10,000 between bovine serum albumin and pituitary neurophysin. The remaining 95% of ovine immunoreactive pineal neurophysin (Mr 10,000) shares immunological and physico-chemical properties with highly purified bovine pituitary neurophysin used as a reference. From the results of gel filtration and affinity chromatography on LVP-Sepharose it was concluded that ovine pineal gland may contain a neurophysin precursor molecule in addition to the neurophysin Mr 10,000.

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.

A high molecular weight fraction XM100R (MW A 100,000) was prepared by ultrafiltration from ovine pineals using two different extraction methods under red light conditions (lambda greater than 600 nm). This fraction stimulates the release of radioimmunologically active luteinizing hormone (LH) of anterior pituitaries in vitro. The ultrafiltration fraction PM30R (MW greater than 30,000 and less than 100,000) was found to be radioimmunologically active only when the "Bensinger" extraction procedure was applied. However, when comparable fractions were prepared under green light and incubated with half-pituitaries, all the incubation media of the ultrafiltrated fractions, XM100R, PM30R, PM10R (MW greater than 10,000 and less than 30,000) UM2R (MW greater than 1000 and less than 10,000), UM05R (MW greater than 500 and less than 1000) and UM05F (MW greater than 500), reacted with anti-LH. This may mean that under green light conditions the high molecular weight ovine pineal compounds in XM100R are disintegrated and/or split up into small molecules which can stimulate the release of LH, or crossreact with the anti-LH serum.

The effect was studied of a number of synthetic indoleamines, pteridines, beta-carbolines, of AVT and of crude extracts from rat and ovine pineal glands on human melanoma cells in vitro. The identified pineal substances as well as some of their analogues showed an inhibitory effect only at non-physiologically high concentrations. However, crude pineal extracts were more active than the synthetic pineal substances tested. They contain a compound which may have a tumor-inhibiting potency comparable to that of methotrexate but a different mechanism of action.

Using rather simple and mild extraction and separation methods, three ovine pineal fractions (XM 300 R-PP7.2' and PP7.2 S) were obtained, which contain peptidic/proteic substances and which show fluorescence characteristics of indoles. The ovine fractions were compared with the bovine pineal E-5 fraction. The ovine fractions are chemically sensitive to normal laboratory light and stable in red light (lambda greater than 600 nm). Immunologically, these fractions and the bovine E 5 fraction are stable. From the results of radioimmunological experiments it was concluded that the bovine pineal E 5 fraction as well as the ovine pineal fraction XM 300 R-PP7.2 and PP7.2S may contain (a) peptide(s) ending by the same carboxy terminal tripeptide Pro-Arg-Gly(NH2).

A bovine pineal acid extract displays a vasotocin-like bioactivity in several bioassays, and is recognized by antibodies against the Pro-Arg-Gly-amide ending common to vasopressin and vasotocin. By using molecular sieve filtration and reversed-phase HPLC, a vasopressin- and oxytocin-like peptide was isolated from this pineal preparation, while no evidence for a vasotocin-like peptide was obtained. The isolated neuropeptides contain a modified amino acid at position 2. This structural difference with authentic pituitary vasopressin and oxytocin may alter their biological and immunological properties, which have been interpreted as vasotocin-like, and thus underlies the controversy concerning the existence of vasotocin in the mammalian pineal gland.

A combination of gelfiltration and reverse-phase high performance liquid chromatography with postcolumn antitumour assay has been developed. A melatonin insensitive human melanoma cell strain was used to guide the purification of the antitumour effect of an ovine pineal aqueous extract (MW 1,000 to 10,000) that possessed the ability to decrease the hypophysiotropic activity of rat and mice hypothalami in vitro. This allows a specific identification of a pineal factor (MW 2,000 to 6,000) that inhibits the growth of human melanoma cells at a dose of 0.47 mg/ml medium. It was shown that the activity of this pineal compound differs from structures known to be present in the pineal, such as melatonin, pteridines, and beta-carbolines. There appears to be evidence for a peptidic nature of this pineal antitumour factor.

High molecular weight substances could be isolated from sheep pineals with the "Bensinger" extraction method, followed by ultrafiltration of the waterlayer through different diaflomembranes. Two of the pineal fractions, XM100R and PM30R, stimulate the gonadotropin releasing activity of the medial basal hypothalamus (MBH). In experiments in which comparable pineal fractions were incubated without MBH and without pituitary and injected in immature mice no effect was detectable. All experiments in which a similar amount of rat cerebral cortex was used for incubation with pineal fractions did not show any activity.

An in vitro human melanoma cell assay was used to work up the partial purification of (a) low molecular weight (MW) substance(s) from aqueous extracts of ovine pineal tissue shown to contain a growth-inhibiting activity. A combination of paper chromatography, ion-exchange and reverse-phase high performance liquid chromatography with post-column antitumor assay has been developed. This allows a specific identification of an ovine pineal factor (MW less than 500) which inhibits the growth of human melanoma cells in vitro. The substance was partially purified to about 1,000 times as compared to the IC100-value of the starting material (retentate 5). The growth inhibition of human melanoma cells in culture was complete at a dose of 0.1 microgram/ml of purified pineal factor(s). It was demonstrated that the activity of this pineal compound differs from some substances known to be present in the pineal, such as melatonin, serotonin, peridines and beta-carbolines. The activity was not destroyed by treatment with proteolytic enzymes.