Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases.

Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.

Identification and characterization of novel uroporphyrinogen decarboxylase gene mutations in a large series of porphyria cutanea tarda patients and relatives.

Porphyria cutanea tarda (PCT) arises from decreased hepatic activity of uroporphyrinogen decarboxylase (UROD). Both genetic and environmental factors interplay in the precipitation of clinically overt PCT, but these factors may vary between different geographic areas. Decreased activity of UROD in erythrocytes was used to identify patients with UROD mutations among a group of 130 Spanish PCT patients. Nineteen patients (14.6%) were found to harbor a mutation in the UROD gene. Eight mutations were novel: M1I, 5del10, A22V, D79N, F84I, Q116X, T141I and Y182C. Five others were previously described: F46L, V134Q, R142Q, P150L and E218G. The new missense mutations and P150L were expressed in Escherichia coli. D79N and P150L resulted in proteins that were localized to inclusion bodies. The other mutations produced recombinant proteins that were purified and showed reduced activity (range: 2.3-73.2% of wild type). These single amino acid changes were predicted to produce complex structural alterations and/or reduced stability of the enzyme. Screening of relatives of the probands showed that 37.5% of mutation carriers demonstrated increased urinary porphyrins. This study emphasizes the role of UROD mutations as a strong risk factor for PCT even in areas where environmental factors (hepatitis C virus) have been shown to be highly associated with the disease.

Evidence for gonadotropin-releasing hormone peptides in the ovary and testis of rainbow trout.

GnRH is usually classified as a neuropeptide that is synthesized in the brain. Recent evidence indicates that GnRH mRNA is present also in the ovary and testis. However, isolation of the peptide from testis has not been reported. We used HPLC and specific RIAs to determine whether the GnRH peptide can be detected in gonads, the developmental stage at which the peptide is expressed, and the number of molecular forms of GnRH that are present in the ovary and testis. Extracts of immature and mature ovarian and testicular tissue were examined from 17- to 21-mo-old rainbow trout (Oncorhynchus mykiss). For the first time, GnRH peptides were isolated from testis and identified by HPLC-RIA with specific antisera and by elution position compared with synthetic standards. GnRH peptides were also present in the ovary. In addition, multiple forms of GnRH, including a form not normally detected in the brain of trout, were shown to be present in the gonads. During development, GnRH peptides were expressed only at specific stages in the gonads, which may explain the inability to detect and isolate the GnRH peptides from gonads in earlier studies.

Gonadotropin-releasing hormone (GnRH) in ancient teleosts, the bonytongue fishes: putative origin of salmon GnRH.

The molecular forms of gonadotropin-releasing hormone (GnRH) were examined in the bonytongue fishes (Osteoglossomorpha), one of the most ancient living teleost groups. These fish represent a phylogenetic link between the early ray-finned fishes and the modern teleosts. Five representative species from four of six bonytongue families were examined for GnRH using high-performance liquid chromatography and radioimmunoassay techniques with antisera raised against salmon (s), chicken-II (c-II), and mammalian (m) forms of GnRH. Salmon GnRH and cGnRH-II were identified in four of the species (arawana, elephantnose, false featherfin, Asiatic featherfin) whereas in the butterfly fish, mGnRH and cGnRH-II were identified. Our data suggest that teleosts such as eels and butterfly fish, which have mGnRH like that of even earlier ray-finned fishes, may have evolved before fish with sGnRH. We also suggest that sGnRH first appeared in the Osteoglossomorpha. The phylogenetic relationship of the eels (Anguillidae), butterfly fish (Pantodontidae), and bonytongue fish among other teleosts needs to be reexamined using additional characteristics.

A second form of gonadotropin-releasing hormone (GnRH) with characteristics of chicken GnRH-II is present in the primate brain.

The primate brain was thought to contain only the GnRH known as mammalian GnRH (mGnRH). This study investigates whether a second form of GnRH exists within the primate brain. We found that brain extracts from adult stumptail and rhesus monkeys contained two forms of GnRH that were similar to mGnRH and chicken GnRH-II (cGnRH-II) based on the elution position of the peptides from HPLC and on cross-reactivity with antisera that are specific to mammalian or chicken GnRH-II in RIAs. The fetal brain of rhesus monkeys also contained mGnRH and a cGnRH-II-like peptide by the same criteria. Immunocytochemistry with a cGnRH-II-specific antiserum in adult and fetal rhesus monkeys showed immunopositive neurons generally scattered in the periaqueductal region of the midbrain, with a few positive cells in the posterior basal hypothalamus. Neurons immunopositive for cGnRH-II were fewer in number and smaller in size, with less defined nuclei and thinner neurites compared with those for mGnRH. Administration of synthetic cGnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration during the luteal phase of the menstrual cycle, but not during the midfollicular phase. We conclude that the primate brain contains mGnRH and a cGnRH-II-like molecule, although the function of the latter is unknown.

Immunoreactive gonadotropin-releasing hormone (GnRH) is detected only in the form of chicken GnRH-II within the brain of the green anole, Anolis carolinensis.

The presence of multiple forms of gonadotropin-releasing hormone (GnRH) within a single brain is common among vertebrate species. In previous studies of reptiles, two forms of GnRH were isolated from the brain of alligators and the primary structure was determined to be that of chicken (c)GnRH-I and cGnRH-II. GnRH has also been detected by indirect methods in other reptiles including turtles, lizards, and snakes. We used a combination of high-performance liquid chromatography (HPLC) and radioimmunoassay to determine the number and molecular form(s) of GnRH in the brain of a lizard, Anolis carolinensis, that was reported to lack GnRH cells in the forebrain. Immunoreactivity was detected in the same HPLC elution position in which synthetic cGnRH-II elutes, but not in any other position. Detection was based on five antisera that among them detect the 12 known forms of GnRH; these antisera include ones that are specific to cGnRH-I and cGnRH-II. We conclude that the lizard A. carolinensis contains cGnRH-II, but not cGnRH-I or another known form of GnRH. These data, coupled with our earlier immunocytochemical study, suggest that the lizard studied here lacks cGnRH-I, the form that is found in the terminal nerve, olfactory bulb, and forebrain in nonsquamate reptiles and in birds. Our hypothesis is that the presence of both cGnRH-I and cGnRH-II in the brain is ancestral in the reptilian lineage and retained in the orders that include turtles (Chelonia) or alligators (Crocodilia). However, the pattern in the order Squamata varies: in A. carolinensis, only cGnRH-II is present in the brain and cGnRH-I is absent, whereas in the snake Thamnophilis sirtalis, cGnRH-I is retained and cGnRH-II is absent in the brain, as recently reported. This raises the question of how reproduction is controlled in reptiles that lack one form of GnRH.

Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain.

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Primary structure of solitary form of gonadotropin-releasing hormone (GnRH) in cichlid pituitary; three forms of GnRH in brain of cichlid and pumpkinseed fish.

GnRH is a decapeptide family with at least nine distinct structures. Vertebrates, except for most placental mammals, have more than one of these GnRH forms within the brain. We report chromatographical and immunological evidence that three forms of GnRH are in the brains of both cichlid (Haplochromis burtoni) and pumpkinseed (Lepomis gibbosus) fishes. We argue that the three forms correspond to those previously described as sea bream GnRH (sbGnRH), chicken GnRH-II and salmon GnRH. In contrast, only one GnRH form was present in the pituitary of the cichlid and is identified as sbGnRH by amino acid sequence. This is the first report in which the primary structure of GnRH is determined from pituitary tissue. The N-terminus was identified by monitoring the digestion of the peptide by pyroglutamate aminopeptidase with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The amidation of the C-terminus was established using an esterification procedure for monitoring with MALDI-MS. This report supports the idea that three forms of GnRH within one species is widespread in the order Perciformes. The present study establishes sbGnRH as the third GnRH form in H. burtoni and predicts that sbGnRH is synthesized in preoptic neurons, then transported to the pituitary in the preoptic-hypophyseal axons for the release of one or both gonadotropins.

Gonadotropin-releasing hormones, including a novel form, in snook Centropomus undecimalis, in comparison with forms in black sea bass Centropristis striata.

The molecular forms of gonadotropin-releasing hormone (GnRH) in brain-pituitary extracts were determined for snook Centropomus undecimalis and black sea bass Centropristis striata. The extracts were analyzed in both isocratic and gradient high performance liquid chromatography (HPLC) programs. Eluted fractions were tested in radioimmunoassays with 4 different antisera made against 3 distinct GnRH peptides. Results show that snook contain 3 forms of GnRH, all of which are present in males and females irrespective of the stage of the reproductive cycle. Larger quantities of these GnRH peptides are present in snook in the nonreproductive phase than in snook in the reproductive phase. One form of snook GnRH is immunologically and chromatographically similar to salmon GnRH, and a second form is similar to chicken GnRH-II. However, the third snook GnRH appears to be distinct from the 7 known forms of the vertebrate hormone. In contrast, sea bass contain only the salmon GnRH-like and chicken GnRH-II-like forms of GnRH and, hence, appear to match the more usual pattern of GnRH peptides in teleosts. We speculate that one of the GnRH genes was duplicated and then altered in a fish ancestral to snook but not sea bass, even though both species of fish are in the recently evolved Perciformes order.

Gonadotropin-releasing hormone from thai catfish: chromatographic and physiological studies.

Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of Thai catfish, Clarias macrocephalus and C. batrachus. The peptides were detected using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). In both the HPLC systems, catfish GnRH-I eluted earlier than catfish GnRH-II and also eluted before the synthetic standards of mammalian, lamprey, chicken I, chicken II, and salmon GnRH. Hence, catfish GnRH-I appears to be the most hydrophilic GnRH family member because of this early elution from the HPLC. Catfish GnRH-II eluted in a position similar to that of chicken GnRH-II. This study suggests that catfish GnRH-I is a novel form of GnRH, whereas catfish GnRH-II is the same as chicken GnRH-II. Indirect evidence suggests that the catfish molecule is 10 amino acids in length and has an amide at the C-terminus. Moreover, the novel catfish GnRH appears to be different within the domain of amino acids 5 to 10 compared with mammalian GnRH because it is not recognized by antiserum B-6. An injection of native chicken GnRH-II was more effective than salmon or mammalian GnRH for induced ovulation in C. macrocephalus.

Mutagenic activity of swimming-pool water.

Swimming pool water, being chlorinated and exposed to trace organics from use was investigated as a possible source of mutagens using the Salmonella/mammalian-microsome test. Procedures previously described for the extraction of trace organics from water using XAD-2 macroreticular resin were modified to allow quantitative extraction of mutagens. These procedures were superior to freeze-drying and solvent-extraction. Using a base-pair histidine mutant, strain TA100, of Salmonella typhimurium significantly mutagenic responses were observed using concentrates from 3 variations of the extraction procedure. Acidified pool-water extracts eluted with ether or acetone were mutagenic, the former enhanced in the presence of the induced microsomal fraction from rat livers. Non-acidified pool-water extracts eluted with acetone were mutagenic without microsomal activation. These results indicate the presence of more than one mutagen in what is likely a complex mixture of organic molecules in swimming-pool water.