Inhibition of prostate cancer cell growth by human secreted PDZ domain-containing protein 2, a potential autocrine prostate tumor suppressor.

A possible role of the PDZ domain-containing protein 2 (PDZD2) in prostate tumorigenesis has been suggested. Besides, PDZD2 is posttranslationally cleaved by a caspase-dependent mechanism to form a secreted PDZ domain-containing protein 2 (sPDZD2) with unknown functions in humans. In this study, we demonstrate the endogenous expression of PDZD2 and secretion of sPDZD2 in cancerous DU145, PC-3, 22Rv1, LNCaP, and immortalized RWPE-1 prostate epithelial cells. Inhibition of endogenous sPDZD2 production and secretion by DU145, PC-3, 22Rv1, and RWPE-1 cells via the caspase-3 inhibitor Z-DEVD-FMK resulted in increased cell proliferation, which was abrogated by treatment with exogenous recombinant sPDZD2. Whereas sPDZD2-induced antiproliferation in DU145, PC-3, and 22Rv1 cells, it induced apoptosis in LNCaP cells. The data suggest that endogenous sPDZD2, produced by caspase-3-mediated cleavage from PDZD2, may function as a novel autocrine growth suppressor for human prostate cancer cells. The antiproliferative effect of sPDZD2 was apparently mediated through slowing the entry of DU145, PC-3, and 22Rv1 cells into the S phase of the cell cycle. In DU145 cells, this can be attributed to stimulated p53 and p21(CIP1/WAF1) expression by sPDZD2. On the other hand, the apoptotic effect of sPDZD2 on LNCaP cells was apparently mediated via p53-independent Bad stimulation. Together our results indicate the presence of p53-dependent and p53-independent PDZD2/sPDZD2 autocrine growth suppressive signaling pathways in human prostate cancer cells and suggest a novel therapeutic approach of harnessing the latent tumor-suppressive potential of an endogenous autocrine signaling protein like sPDZD2 to inhibit prostate cancer growth.

Louping ill virus envelope protein expressed by recombinant baculovirus and vaccinia virus fails to stimulate protective immunity.

We have constructed recombinant baculoviruses and vaccinia viruses containing cloned DNA, encoding either the envelope protein alone or all of the structural proteins (core, membrane and envelope) of louping ill virus. Glycosylated viral envelope protein, presented both inside and on the surface of insect and mammalian cells, was expressed by all four recombinant viruses. Differences in antigenic presentation of the envelope protein were observed between the envelope protein and structural protein constructs as well as between the insect and mammalian cell expression systems. Despite the expression of epitopes known to elicit neutralizing and protective antibodies when present in authentic antigen, the recombinant envelope protein expressed by either vector failed to induce, in mice or rabbits, either neutralizing or protective antibodies against louping ill virus.

Neuroendocrinology of melatonin in reproduction: recent developments.

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

Cold exposure or norepinephrine injections altered melatonin levels in the quail retina.

The possible involvement of ambient temperature and norepinephrine on daytime levels of retinal melatonin were investigated in quails. For a minimum of 1 week, experimental animals were housed under constant room temperature of 23 +/- 2 degrees C and a daily 12:12 h light-dark cycle with light on at 06.00-18.00 h. The quails were then transferred to a cold room of 4 degrees C and cold-exposed for 0, 30, 60, 90 and 120 min. Retina samples were subsequently collected at mid-light for melatonin radioimmunoassay. An initial decline of melatonin was detected in the cold treated birds after 30 min of exposure. Thereafter prolongation of cold stimulation produced significant increases in the levels of retinal melatonin. In the second experiment, intra-peritoneal norepinephrine injections (0, 1, 10 and 100 micrograms/bird) at mid-light were found to increase the levels of retinal melatonin in quails. We postulate the cold-induced increase of retinal melatonin may be attributed to an augmented level of catecholamines released as a general neuroendocrine response to temperature decrements.

Melatonin-induced stimulation of rat corpus epididymal epithelial cell proliferation.

Stimulation of rat epididymal epithelial cell proliferation by melatonin was demonstrated by thymidine incorporation and flow cytometric analyses. The stimulatory effect of melatonin was dependent on the hormone concentration and the duration of cell exposure to the hormone. Maximal stimulation of [3H]thymidine incorporation into epididymal epithelial cells by melatonin was observed at 1 x 10(-9) M 5alpha-dihydrotestosterone in medium, while lower or higher concentrations of androgen attenuated the stimulatory effect of melatonin. Interestingly, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxothiazolidine-2-ylidene]-4-methyl-thiosemi-carb azone, CGP 52608) induced opposite effect on epithelial cell proliferation to that produced by melatonin. Our data suggest that melatonin-induced stimulation of rat epididymal epithelial cell proliferation is not likely to be mediated by nuclear receptor. Furthermore, sequential changes of cell cycle distribution with melatonin treatment also supports a stimulatory action of melatonin on epididymal epithelial cell proliferation.

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with beta-amyloid 1-40.

Intraventricular infusion of rats with beta-amyloid for 14 days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus. These changes were not altered by daily intraperitoneal injection of 20 mg/Kg melatonin. Orally administered Ginkgo biloba extract, however, partially reversed the memory deficit and the decrease in choline actyltransferase activities in the hippocampus. The latter treatment failed to reverse the decrease in somatostatin levels. The results indicate that orally administered Ginkgo biloba extract can protect the brain against beta-amyloid from changes leading to memory deficit through its effect on the cholinergic system.

Autoradiographic distribution and physiological regulation of 2-[125I]iodomelatonin binding in rat epididymis.

Autoradiographic study was conducted to localize 2-[125I]iodomelatonin binding in the rat epididymis. In the peripubertal (6 weeks old), postpubertal (8 weeks old) and adult (3 months old) rats, intense specific 2-[125I]iodomelatonin labelling of the corpus epididymidis was observed. The intensity of 2-[125I]iodomelatonin binding in the distal epididymal segment was significantly decreased in orchidectomized rats but the effect could be reversed with testosterone replacement. The intensity of 2-[125I]iodomelatonin binding in the distal rat epididymal segment did not show any diurnal rhythmicity when mid-light period and mid-dark period levels were compared, and was unaffected by constant lighting. Our data suggest androgen-dependent expression of 2-[125I]iodomelatonin binding sites, independent of light-induced changes in circulating melatonin, in the rat corpus epididymidis. A novel role of melatonin and its receptor in the regulation of the functions of rat corpus epididymidis is strongly implicated.

Inhibition of malignant trophoblastic cell proliferation in vitro and in vivo by melatonin.

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT2 receptor, but not that of mt1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

Association between polyomaviruria and microscopic haematuria in bone marrow transplant recipients.

The association of polyomaviruria and microscopic haematuria was studied by the use of electron microscopy (EM) and the polymerase chain reaction (PCR) in bone marrow transplant (BMT) recipients. The incidence of BK virus (BKV) and JC virus (JCV) excretion was further elucidated by means of restriction enzyme analysis of the PCR products. Polyomaviruses were detected in 43 (51.2%) of the 84 samples, 13 (30.2%) of which had a virus concentration detectable by EM. By typing with BamHI cleavage, 29 (67.4%) of the 43 positive patients were found to be excreting only BKV and the remaining 14% (32.6%) were excreting both BKV and JCV. Microscopic haematuria was present in 17 (20.2%) of 84 urine samples collected from different patients within 4 months post-transplant. The incidence of microscopic haematuria was significantly higher, 34.9% (P < 0.01), in patients with polyomaviruria than in those without (4.9%) but no difference was observed between the BKV-excreting and BKV/JCV-co-excreting patients. Microscopic haematuria was not present, however, in 53.8 and 65.2% of polyomavirus-excreting patients when virus was detected by EM and PCR respectively. While most episodes of microscopic haematuria observed were self-limiting and asymptomatic, three patients excreting polyomavirus had symptoms of cystitis and one of them had renal impairment that was otherwise unexplained. We thus conclude that polyomaviruses probably contribute to damage of urinary tract tissue in some BMT recipients.

Recombinant vaccinia virus expressing PrM and E glycoproteins of louping ill virus: induction of partial homologous and heterologous protection in mice.

Recombinant vaccinia viruses expressing either the premembrane/truncated envelope (PrM/TrE) or truncated envelope (TrE) protein of louping ill virus were constructed. Both constructs expressed authentic E proteins as determined by their size and antigenic reactivity with a panel of monoclonal antibodies. The deletion of the C-terminal hydrophobic domain of the envelope glycoprotein resulted in the secretion of E protein into the supernatant culture medium. The immunisation of mice with these recombinant viruses showed that the recombinant expressing PrM/TrE proteins induced neutralising and protective antibodies against challenge with louping ill or tick-borne encephalitis virus, but that the recombinant expressing the E or the TrE protein alone failed to induce any detectable immune responses against homologous or heterologous virus challenge.

Melatonin: a chemical photoperiodic signal with clinical significance in humans.

Secretion of pineal melatonin exhibits a diumal rhythm and a seasonal rhythm in humans. Night-time melatonin is high at 3-5 year-old and decreases with age. Many drugs and pathological conditions also change melatonin levels in the circulation. Melatonin has a mild sedative effect and has been used effectively in synchronizing the sleep-wake cycle of patients with sleep disorders. Immunoenhancing, anti-cancer, anti-aging and anti-oxidant effects of melatonin have been proposed. Recent studies suggest that melatonin receptors are present in central and peripheral tissues. The importance of melatonin receptors on the nervous, reproductive, immune and renal functions is implicated. Studies on the molecular biology, physiology and pathology of melatonin receptors in different tissues are progressing rapidly. The physiological and pathological changes in melatonin secretion, multifarious melatonin actions, and diverse melatonin receptors reported suggest that melatonin is a photoperiodic signal with clinical significance in humans.

An updated phylogenetic analysis of vertebrate melatonin receptor sequences: reflection on the melatonin receptor nomenclature by the Nomenclature Subcommittee of the International Union of Pharmacology.

In the past few years, significant progress on melatonin receptor research has led to the discovery of a family of genetically related but pharmacologically distinctive G-protein-coupled receptors in the vertebrates. With increasing number of receptor clones being identified, there is a need for a system of classification and nomenclature for these receptor subtypes. Recently, an updated nomenclature system, which has renamed the existing mammalian melatonin receptor clones, has been proposed by the relevant subcommittee of the International Union of Pharmacology (NC-IUPHAR). However, the majority of receptor clones which have been identified in non-mammalian vertebrates are not clearly defined by this system. By performing phylogenetic analysis of both mammalian and non-mammalian melatonin receptor clones, we would like to propose a classification-nomenclature system for vertebrate melatonin receptors. Hopefully, our system, which incorporates genetic data as well as the pharmacological criteria that have been adopted by the NC-IUPHAR nomenclature system, will provide the framework for future development of a unified scheme of classification and nomenclature for melatonin receptors.

A molecular perspective of the genetic relationships of G-protein coupled melatonin receptor subtypes.

Successful cloning of melatonin receptors from various target tissues in the past few years has increased our understanding of the molecular signal transduction mechanisms of G-protein coupled melatonin receptors, of which three subtypes (MEL-1A, MEL-1B, and MEL-1C) have been reported in different vertebrates. Based upon melatonin receptor sequences available in the Genbank database, we have performed phylogenetic analyses of the nucleotide and encoded amino acid sequences of G-protein-coupled melatonin receptors, and determined the range of amino acid identities between melatonin receptors of the same and different subtypes. Besides the three well-known subtypes, a potential novel subtype of MEL-1D, as exemplified by unique separation of Xenopus X2.0 sequence (Genbank accession No. U31826) from the others in the protein phylogenetic tree, possibly exists. In addition, one of the chicken brain melatonin receptor sequences has been identified as belonging to the MEL-1B subtype. Our analyses showed that melatonin receptors of the same subtype and different subtypes are likely to share > or = 75% and < 65% amino acid identities, respectively. Phylogenetic analysis based on amino acid comparisons will be needed to determine the subtype status of any pair of melatonin receptor sequences that exhibit > or = 65% to < 75% amino acid identity. Despite the usefulness of genetic relatedness in the subtype classification of G-protein-coupled melatonin receptors, functional correlation of molecular structure may ultimately prove the most comprehensive approach in melatonin receptor classification.

Genomic sequence of the structural proteins of louping ill virus: comparative analysis with tick-borne encephalitis virus.

The genomic RNA of louping ill virus coding for capsid, premembrane, membrane, and envelope proteins was cloned and sequenced. Hydrophilicity profiles of the deduced amino acid sequence shared homologous functional domains with other flaviviruses. The premembrane and envelope proteins contain N-glycosylation sites and conserved cysteine residues which are important for maintaining the secondary structures of the proteins. Sequence comparisons of louping ill envelope protein showed greater homology with tick-borne than mosquito-borne flaviviruses and greater homology with the western than the far eastern subtype of tick-borne encephalitis virus. With the capsid and membrane proteins, the degree of homology between louping ill and the western subtype was greater than that between the two subtypes, indicating very close evolutionary relationships between louping ill and the western subtype of tick-borne encephalitis. Thus, louping ill and tick-borne encephalitis may be varieties of a common tick-borne ancestral virus. The average amino acid sequence diversity between members of the tick-borne serogroup was significantly lower than that of mosquito-borne serogroups, suggesting that tick-borne flaviviruses have been subjected to different evolutionary immune selection pressure from the mosquito-borne viruses. Using the published model of tick-borne encephalitis envelope protein and our sequence data on louping ill virus, we have identified three discontinuous peptides (amino acids 81-88, 207-212, and 230-234) which may represent critical molecular determinants within the receptor binding site of tick-borne flaviviruses and may provide a specific genetic marker for these viruses.

Effect of photic manipulation on the level of melatonin in the retinas of frogs (Rana tigrina regulosa).

The level of melatonin in the frog (Rana tigrina regulosa) retina was studied at midlight and middark in a 12L:12D cycle and under different lighting conditions. It was found that the frog displayed a diurnal rhythm of melatonin in the retina with high levels in the dark period. When the animal was subjected to an extended dark period, the level of retinal melatonin was significantly (P less than 0.05) increased. In addition, the normal low level of retinal melatonin in the light period was significantly (P less than 0.05) increased after dark treatment, and the normal high level of retinal melatonin in the dark period was significantly (P less than 0.05) lowered following light exposure. These results suggest that synthesis and secretion of melatonin in the frog retina is controlled by environmental lighting. This supports the hypothesis that melatonin plays an important role in the regulation of the photomechanical changes of eye pigmentation, an important element in the control of light sensitivity and acuity in the eyes of vertebrates. Moreover, these findings suggest that the photoreceptor may be a neuroendocrine or neurohumoral transducer which transduces the environmental lighting into neuroendocrine or neurohumoral secretion, melatonin.

Comparative analysis of the NS 1 gene sequences of dengue-1 viruses prototype Hawaii strain and Thai isolate TH-Sman, and determination of the intratypic variation of NS 1 protein among dengue-1 viruses.

In view of a previous report on significant antigenic and biophysical differences between the purified soluble complement-fixing antigens of dengue-1 virus strains Hawaii and TH-Sman, the NS 1 genes of both virus isolates were cloned, sequenced, and compared in an attempt to define the genetic basis for the observed differences. Sequence comparison revealed ten encoded amino acid differences between the NS 1 genes of both viruses. Three of these amino acid differences, which are associated with a change in charge distribution, are clustered within the major antigenic region previously defined by studies of recombinant dengue-1 NS 1 protein expressed in E. coli. In parallel, the NS 1 sequences of both Hawaii and TH-Sman isolates were also aligned and compared with two other published dengue-1 NS 1 protein sequences to determine the intratypic variation of dengue-1 NS 1 antigen. Pairwise comparisons between the encoded amino acid sequences revealed a variability of 1.1% to 3.1% difference in the NS 1 protein among dengue-1 strains, which is comparable to that reported for dengue-1 envelope protein (0.2% to 3.6% difference) but less than that of dengue-2 NS 1 protein (0.6% to 7.4% difference).

Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses.

Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.

Regional and diurnal studies of melatonin and melatonin binding sites in the duck gastro-intestinal tract.

Since melatonin and putative melatonin receptors can be detected in a variety of peripheral tissues, direct endocrine and paracrine actions of melatonin on the physiological functions of different organ systems in response to internal and external stimuli probably exist. As an extension of our earlier work on the pharmacological characterization of 2-[125I]iodomelatonin binding sites in the duck jejunum, the regional and diurnal variations of melatonin and putative melatonin receptors of different segments of the duck gastro-intestinal tract were studied in an attempt to understand the role of melatonin in the physiology of the digestive system. Although no significant effects of diurnal variation and pinealectomy on the regional distribution of melatonin were observed, significant regional variations of melatonin levels were detected with decreasing levels as follows: colon > oesophagus, caecum > duodenum, jejunum > ileum. The densities of melatonin binding sites showed a significant variation between different intestinal regions at either mid-light or mid-dark, with the following descending order: ileum, jejunum > duodenum, colon > caecum > oesophagus. Analysis of the distribution of melatonin binding sites in the wall of the intestine revealed maximal binding in the mucosa and minimal binding in the muscular layers of the jejunum. Similar results were obtained for other intestinal regions as revealed by autoradiography. No significant changes in the affinities of melatonin binding sites were detected between different regions and tissue layers of the alimentary canal. Moreover, the densities and affinities of melatonin binding sites among different regions of the gut exhibited no significant diurnal variations. The demonstration of regional variations in melatonin levels and the density of melatonin binding sites along the gastro-intestinal tract, with a concentration of the putative melatonin receptors in the mucosal layer, suggests a possible direct action of melatonin in the regulation of fluid/electrolyte transport and nutrient absorption in the gut.

Inhibition of androgen-sensitive LNCaP prostate cancer growth in vivo by melatonin: association of antiproliferative action of the pineal hormone with mt1 receptor protein expression.

BACKGROUND: Potential involvement of the mt1 receptor in the antiproliferative action of melatonin on androgen-sensitive LNCaP cells, and melatonin-induced modulation of androgen-insensitive PC-3 cell growth, have been reported in vitro. The effects of melatonin on prostate cancer cell proliferation and their association with mt1 receptor expression were investigated in athymic nude mice xenograft models of LNCaP and PC-3 cells. METHODS: Daily saline or melatonin (4 microg/g body weight) was given to nude mice before or after tumor cell inoculation. Tumor volume was measured periodically, and expression of PCNA, cyclin A, PSA, and mt1 receptor was assessed by immunohisto(cyto)chemistry and/or Western blotting. RESULTS: Melatonin inhibited the growth of LNCaP tumors, without affecting the growth of PC-3 xenografts, in nude mice. It induced significant decreases in the expression of PCNA, cyclin A, and PSA in LNCaP tumors. Expression of mt1 receptor protein was demonstrated in LNCaP cells, but not in PC-3 cells, both in vivo and in vitro. CONCLUSIONS: The antiproliferative action of melatonin on LNCaP tumor growth was demonstrated in vivo, and its association with mt1 receptor protein expression suggests the potential involvement of the receptor in the antitumor activity of the pineal gland hormone.