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.

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.

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.

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.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells.

Melatonin, a pineal secretory product, has been shown to exert a direct anti-proliferative action on the androgen-sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using 3H-thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2-iodomelatonin, a high-affinity melatonin receptor agonist. Furthermore, melatonin inhibited 3H-thymidine incorporation into LNCaP cells and attenuated 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2)-induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration-dependent inhibition of sex steroid-induced stimulation of thymidine incorporation into LNCaP cells by 2-iodomelatonin was also observed. Interestingly, attenuation of sex steroid-stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2-iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate-specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid-induced calcium influx are two important mechanisms mediating the direct anti-proliferative action of melatonin on androgen-responsive human prostate cancer cells.

Biological basis and possible physiological implications of melatonin receptor-mediated signaling in the rat epididymis.

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[(125)I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[(125)I]iodomelatonin binding sites to be mt(1) (MEL(1A)) and MT(2) (MEL(1B)) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive G(i) protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT(2) receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-car bazone , CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt(1) and MT(2) melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.

Melatonin-induced inhibition of proliferation and G1/S cell cycle transition delay of human choriocarcinoma JAr cells: possible involvement of MT2 (MEL1B) receptor.

Melatonin, the pineal neurohormone, is an evolutionarily conserved photoperiodic signaling molecule with diverse functions that include the entrainment of human circadian rhythms. Although evidence supporting a direct inhibitory action of melatonin on human cancer cell proliferation exists in the literature, the molecular and cellular signaling mechanisms involved are largely undefined. In our study, significant inhibition of human choriocarcinoma JAr cell proliferation at physiological and pharmacological concentrations of melatonin was observed. 2-Iodomelatonin, a high affinity melatonin receptor agonist, was more potent than melatonin in inhibiting JAr cell proliferation. In addition, the presence of putative melatonin receptors in choriocarcinoma was suggested by the demonstration of specific 2-[125I]iodomelatonin binding to the tumor. Interestingly, the selective MT2 melatonin receptor ligand, 4-phenyl-2-propionamidotetraline (4-P-PDOT), was found to exert not only concentration-dependent anti-proliferative actions on JAr cells, but also additive effects with melatonin in inhibiting JAr cell proliferation. Furthermore, MT2 melatonin receptor gene expression by JAr cells was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). Taken together, our data suggest that the reported anti-proliferative action of melatonin on human choriocarcinoma JAr cells may be mediated, in part, by MT2 melatonin receptor. Moreover, analysis of melatonin effect on cell cycle kinetics indicated that G1/S transition delay may underlie the observed inhibition of choriocarcinoma cell proliferation by melatonin.

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.

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.

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.

Molecular and cellular analyses of melatonin receptor-mediated cAMP signaling in rat corpus epididymis.

By using 2-[125I]iodomelatonin receptor binding studies, we have previously demonstrated high affinity melatonin receptors, the binding activities of which are regulated by testosterone, in the corpus epididymis of rats. In this report, some of the basic molecular and cellular characteristics of these high affinity melatonin receptors in rat corpus epididymis were analyzed. MEL1A and MEL1B receptor mRNAs were expressed by rat corpus epididymal epithelial cells as revealed by in situ hybridization. Functionally, these high affinity melatonin receptors are negatively coupled to adenylyl cyclase via pertussis toxin (PTX) sensitive Gi protein and the inhibitory effects of melatonin on forskolin-stimulated cAMP accumulation were enhanced by 5alpha-dihydrotestosterone (5alpha-DHT). Interestingly, opposing interactions between melatonin and beta-adrenergic receptor signaling in rat epididymal epithelial cells were observed with melatonin inhibiting norepinephrine- and isoproterenol-stimulated cAMP accumulation. In conclusion, our data support a modulatory action of melatonin, mediated via pertussis toxin-sensitive Gi-coupled MEL1A and MEL1B receptors, in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions.

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.

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.

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.

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.

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.

Sequence variation of the amino-terminal antigenic domains of glycoprotein B of human cytomegalovirus strains isolated from Chinese patients.

To study the genetic variation of human cytomegalovirus (HCMV) in Asian populations, the amino-terminal antigenic domains of glycoprotein B of HCMVs isolated from ethnic Chinese transplant patients were cloned and sequenced. The nucleotide and encoded amino acid sequences were compared with published sequences of AD169 and Towne laboratory strains. Within the region sequenced, 9 out of 15 clinical isolates (60%) possessed a peptide configuration similar to that of strain AD169 while 6 isolates (40%) displayed a peptide configuration similar to that of strain Towne. The nucleotide and amino acid identities of AD169-like clinical isolates exhibited variations of 95.4%-99.6% and 95.4%-100% respectively, whereas the identities of Towne-like clinical isolates were within the range of 97.3%-100% and 96.6%-100% at the nucleotide and amino acid levels. The previously defined neutralizing epitope was conserved among the clinical isolates sequenced while unique non-conservative amino acid substitutions were detected in the non-neutralizing epitope within the amino-terminal antigenic domain of glycoprotein B of all AD169-like isolates (Y- > S) and one of the Towne-like isolates (R- > Q).

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).