Spatial distribution and fruiting phenology of Protium heptaphyllum (Burseraceae) determine the design of the underground foraging system of Atta sexdens L. (Hymenoptera: Formicidae).

Leaf-cutting ants have long been recognized to forage via complex trail systems but the nature and the ecological drivers of the different foraging strategies adopted remain a key topic. Here, we described the spatiotemporal use of belowground foraging galleries by Atta sexdens L. in the Brazilian Atlantic forest, and examined the adaptive advantages of this foraging strategy. Protium heptaphyllum adult trees (DBH > 10 cm), seed/seedling clumps and ant gallery entrances were mapped across two 1-ha plots during two consecutive fruiting seasons (2002 and 2004). We recorded 75 ca. 40 cm deep gallery entrances beneath 26 P. heptaphyllum trees at nest distances ranging from 14 to 57 m. Furthermore, gallery abundance and galleries associated with seed/seedling clumps correlated positively with P. heptaphyllum density. Our results indicate that A. sexdens was able to set a permanent system of underground galleries targeting P. heptaphyllum trees and their seeds on the ground. Such network of galleries was spatially arranged according to both the spatial distribution and abundance of P. heptaphyllum trees in a way that most gallery entrances were disposed beneath or in close periphery of P. heptaphyllum crowns. Our findings suggest that underground trail systems shaped by fruit resources represent a foraging strategy clearly more common than existing literature on the subject would suggest. In addition, it reinforces the notion that the spatiotemporal availability of resources combined with predation risk largely influence trail configurations as well as overall foraging strategies adopted by leaf-cutting ants.

Relocation of Croton sonderianus (Euphorbiaceae) seeds by Pheidole fallax Mayr (Formicidae): a case of post-dispersal seed protection by ants?

Although seed dispersal by ants might reduce seed predation near the parent plants, predation on discarded seeds clustered on nest refuse piles may reduce any initial benefit provided by seed removal. Here we examine the fate of Croton sonderianus seeds that were discarded by Pheidole fallax Mayr ants on their nest refuses in caatinga vegetation of northeast Brazil. We collected all seeds discarded in refuse piles of 20 nests and within a radius of 50 cm from their borders, and examined them for evidence of predation. A total of 3,017 seeds were recorded either located in the P. fallax refuse piles (89.1%) or nest vicinity (10.9%). Predation was three fold higher in nest vicinity as compared to refuse piles. By removing seeds from beneath parent plants and relocating then to refuse piles, P. fallax is possibly providing double protection services for C. sonderianus seeds. Our findings represent the first evidence for predator-avoidance as benefit for plants resulting from ant seed-dispersal in the neotropics.

Relocation of Croton sonderianus (Euphorbiaceae) seeds by Pheidole fallax Mayr (Formicidae): a case of post-dispersal seed protection by ants?

Although seed dispersal by ants might reduce seed predation near the parent plants, predation on discarded seeds clustered on nest refuse piles may reduce any initial benefit provided by seed removal. Here we examine the fate of Croton sonderianus seeds that were discarded by Pheidole fallax Mayr ants on their nest refuses in caatinga vegetation of northeast Brazil. We collected all seeds discarded in refuse piles of 20 nests and within a radius of 50 cm from their borders, and examined them for evidence of predation. A total of 3,017 seeds were recorded either located in the P. fallax refuse piles (89.1 percent) or nest vicinity (10.9 percent). Predation was three fold higher in nest vicinity as compared to refuse piles. By removing seeds from beneath parent plants and relocating then to refuse piles, P. fallax is possibly providing double protection services for C. sonderianus seeds. Our findings represent the first evidence for predator-avoidance as benefit for plants resulting from ant seed-dispersal in the neotropics.

Seed predation by rodents and safe sites for large-seeded trees in a fragment of the Brazilian Atlantic forest.

Seed predation by small rodents is an emerging theme in the ecology of modified landscapes. Here we investigate the role played by the small rodent Oryzomys oniscus as a seed predator of large-seeded trees in a large remnant of the Atlantic forest - the Coimbra forest (3,500 ha), Alagoas state, northeastern Brazil. O. oniscus was captured and identified by setting twenty 500 m long transects, each one composed of 25 traps 20 m apart. This procedure resulted in 483 trap-nights set during a 20-mo period. We used 692 seeds (>15 mm length) from ten local large-seeded tree species for the seed predation trials that basically consisted of three treatments: one seed on the ground freely accessed by vertebrates (unprotected seed), one seed totally protected by an exclosure, and one partially-protected seed (exclosure just for medium-sized and large vertebrates). O. oniscus was captured throughout the Coimbra forest including forest edges (76 captures) and interior areas (67), and this small rodent was responsible for all seed predation visually documented inside exclosures. A 24 hours period of seed exposition permitted elevated rates of seed removal and predation. Seeds were much more removed/predated beneath fruiting trees, but rates varied according to the level of seed protection--26.3% of predation among partially-protected versus 19.2% among unprotected seeds. Seeds suffered higher levels of seed predation/removal at the forest edge as well (up to 90%). In both habitats, most seeds (>84%) remained intact beneath trees without fruits, regardless of the level of seed protection. Our results suggest that O. oniscus may operate as an effective large-seed predator in forest fragments, in which adult trees without fruits constitute low resource spots and thereby provide, at least temporarily, safe sites for large seeds.

Seed predation by rodents and safe sites for large-seeded trees in a fragment of the Brazilian Atlantic forest / Predação de sementes por roedores e sítios seguros para árvores com grandes sementes em um fragmento de floresta Atlântica brasileira

Seed predation by small rodents is an emerging theme in the ecology of modified landscapes. Here we investigate the role played by the small rodent Oryzomys oniscus as a seed predator of large-seeded trees in a large remnant of the Atlantic forest - the Coimbra forest (3,500 ha), Alagoas state, northeastern Brazil. O. oniscus was captured and identified by setting twenty 500 m long transects, each one composed of 25 traps 20 m apart. This procedure resulted in 483 trap-nights set during a 20-mo period. We used 692 seeds (>15 mm length) from ten local large-seeded tree species for the seed predation trials that basically consisted of three treatments: one seed on the ground freely accessed by vertebrates (unprotected seed), one seed totally protected by an exclosure, and one partially-protected seed (exclosure just for medium-sized and large vertebrates). O. oniscus was captured throughout the Coimbra forest including forest edges (76 captures) and interior areas (67), and this small rodent was responsible for all seed predation visually documented inside exclosures. A 24 hours period of seed exposition permitted elevated rates of seed removal and predation. Seeds were much more removed/predated beneath fruiting trees, but rates varied according to the level of seed protection - 26.3 percent of predation among partially-protected versus 19.2 percent among unprotected seeds. Seeds suffered higher levels of seed predation/removal at the forest edge as well (up to 90 percent). In both habitats, most seeds (>84 percent) remained intact beneath trees without fruits, regardless of the level of seed protection. Our results suggest that O. oniscus may operate as an effective large-seed predator in forest fragments, in which adult trees without fruits constitute low resource spots and thereby provide, at least temporarily, safe sites for large seeds.

Predação de sementes por pequenos roedores é um tema emergente na ecologia de paisagens modificadas. Nesse estudo foi investigado o papel desempenhado pelo pequeno roedor Oryzomys oniscus como predador de grandes sementes de árvores em um grande fragmento de floresta Atlântica - mata de Coimbra (3.500 ha), Estado de Alagoas, Nordeste do Brasil. O. oniscus foi capturado e identificado usando-se 20 transectos com 500 m de comprimento, cada um composto por 25 armadilhas espaçadas a cada 20 m. Esse procedimento resultou em um esforço total de captura de 483 armadilhas-noites, durante um período de 20 meses. Foram utilizadas 692 grandes sementes (>15 mm de comprimento), provenientes de dez espécies de árvores para as sessões de predação, as quais consistiram basicamente de três tratamentos: uma semente no solo com acesso livre para vertebrados (semente desprotegida), uma semente totalmente protegida e uma semente parcialmente protegida (exclusão apenas para médios e grandes vertebrados). O. oniscus foi capturado em todo o fragmento de Coimbra, incluindo bordas de floresta (76 capturas) e áreas de interior (67 capturas). Além disso, esse pequeno roedor foi responsável por toda a predação de sementes visualmente documentada dentro das gaiolas de proteção de sementes. Um período de 24 horas de exposição das sementes permitiu elevadas taxas de remoção e predação. Sementes foram muito mais predadas/removidas debaixo de árvores frutificando, mas as taxas variaram de acordo com o nível de proteção das sementes - 26,3 por cento de predação entre as parcialmente protegidas versus 19,2 por cento entre sementes não protegidas. Sementes sofreram altos níveis de predação/remoção nas bordas de florestas (até 90 por cento). Em ambos os hábitats, muitas sementes (>84 por cento) permaneceram intactas debaixo de árvores sem frutos, independente do nível de proteção. Os resultados do estudo sugerem que O. oniscus pode agir como um predador efetivo de grandes ...

Seed shadow, seedling recruitment, and spatial distribution of Buchenavia capitata (Combretaceae) in a fragment of the Brazilian Atlantic forest.

Here we describe the seed shadow, seedling recruitment, ontogenetic structure and spatial distribution of Buchenavia capitata (an emergent canopy tree) in a 380-ha fragment of the Atlantic forest in northeast Brazil. In particular, we examine seed distribution around 10 parental trees and both seedling recruitment and mortality, during an 18 month period beneath and around parental trees. Moreover, we describe: (1) B. capitata occurrence within treefall gaps; (2) population structure in terms of ontogenetic stages for the whole site; and (3) spatial distribution of adults within an area of 51 hectares. 99% of seeds were found beneath parent crowns (n = 4,236) and seed density reached 14.6 +/- 29.9 seeds/m2 (0-140 seeds/m2). 49% of all seeds germinated but seedling mortality reached 100% after an 18 month period. In addition, saplings of B. capitata were not found in forest understory and within 30 treefall gaps (94-2,350 m2). The adults showed an average DBH of 69.3 +/- 22.1 cm, were 19.2 +/- 2.9 m tall and presented a clumped spatial distribution. B. capitata matched some of the features presented by shade intolerant trees or large-gap specialists, and we hypothesize that low rates or even lack of long distance seed dispersal events may be reducing the probability of B. capitata seeds reaching suitable habitats for successful seedling recruitment and growth. Because of that (1) seedlings face high levels of early mortality; (2) there is no sapling recruitment at the study site; and (3) local population faces senility and it is threatened by local extinction.

Seed shadow, seedling recruitment, and spatial distribution of Buchenavia capitata (Combretaceae) in a fragment of the Brazilian Atlantic Forest / Sombra de sementes, recrutamento de plântulas e distribuição espacial de Buchenavia capitata em um fragmento da Floresta Atlântica Brasileira

Here we describe the seed shadow, seedling recruitment, ontogenetic structure and spatial distribution of Buchenavia capitata (an emergent canopy tree) in a 380-ha fragment of the Atlantic forest in northeast Brazil. In particular, we examine seed distribution around 10 parental trees and both seedling recruitment and mortality, during an 18 month period beneath and around parental trees. Moreover, we describe: (1) B. capitata occurrence within treefall gaps; (2) population structure in terms of ontogenetic stages for the whole site; and (3) spatial distribution of adults within an area of 51 hectares. 99 percent of seeds were found beneath parent crowns (n = 4,236) and seed density reached 14.6 ± 29.9 seeds/m² (0-140 seeds/m²). 49 percent of all seeds germinated but seedling mortality reached 100 percent after an 18 month period. In addition, saplings of B. capitata were not found in forest understory and within 30 treefall gaps (94-2,350 m²). The adults showed an average DBH of 69.3 ± 22.1 cm, were 19.2 ± 2.9 m tall and presented a clumped spatial distribution. B. capitata matched some of the features presented by shade intolerant trees or large-gap specialists, and we hypothesize that low rates or even lack of long distance seed dispersal events may be reducing the probability of B. capitata seeds reaching suitable habitats for successful seedling recruitment and growth. Because of that (1) seedlings face high levels of early mortality; (2) there is no sapling recruitment at the study site; and (3) local population faces senility and it is threatened by local extinction.

Neste estudo, são descritas a sombra de sementes, o recrutamento de plântulas, a estrutura ontogenética e a distribuição espacial de Buchenavia capitata (árvore emergente) em um fragmento de 380 ha da floresta Atlântica do Nordeste do Brasil. Particularmente, são examinadas a distribuição de sementes de 10 árvores e o recrutamento e a mortalidade de plântulas durante 18 meses, tanto embaixo quanto em volta dos adultos. Além disso, descrevem-se: (1) a ocorrência de B. capitata em 30 clareiras; (2) a estrutura populacional em termos de estágios ontogenéticos para toda a área; e (3) a distribuição espacial dos adultos dentro de uma área de 51 hectares. Noventa e nove por cento das sementes foram encontradas embaixo das copas das matrizes (n = 4236) e a densidade de sementes alcançou 14,6 ± 29,9 sementes/m² (0-140 sementes/m²). Quarenta e nove por cento de todas as sementes germinaram, mas a mortalidade de plântulas atingiu 100 por cento após 18 meses. Além disso, jovens de B. capitata não foram encontrados no sub-bosque e em 30 clareiras (94-2350 m²). Os adultos mostraram um DAP médio de 69,3 ± 22,1 cm, 19,2 ± 2,9 m de altura e apresentaram uma distribuição espacial agregada. B. capitata apresenta características comuns às árvores intolerantes à sombra ou especialistas de grandes clareiras. Baixas taxas, ou mesmo a ausência, de dispersão de sementes a longa distância podem estar reduzindo a probabilidade de sementes de B. capitata alcançarem hábitats adequados ao recrutamento e ao desenvolvimento das plântulas. Por causa disto (1) as plântulas enfrentam altos níveis de mortalidade; (2) não tem ocorrido recrutamento de jovens; e (3) a população é senil e está ameaçada de extinção local.

Distance from roads and cities as a predictor of habitat loss and fragmentation in the caatinga vegetation of Brazil

Roads and cities represent huge sources of degradation for adjacent ecosystems regarding nutrient cycling, energy, water flow and species composition. In this study we test the hypothesis that distance from roads and cities is associated with habitat loss and fragmentation in the caatinga vegetation- a dry forest to scrub vegetation that covers ca. 736,000 km² of northeast Brazil. The study site comprised a 2,828.8 km² piece (64 km x 44.2 km) of Xingó region (09°36'S, 37°50'W), which is located between the States of Alagoas and Sergipe. Based on satellite imagery we mapped the remaining vegetation, 145 km of paved roads and the seven small-sized cities set in the study site. A positive correlation was found between the combined distance from roads and cities and the percentage of remaining vegetation as it dropped from 18 percent at 12 km distant to 5.9 percent at 1 km distant from cities and roads. Thus, remaining vegetation was reduced by one third near cities and roads. A positive correlation was also found between distance from cities and roads and the percentage of fragments larger than 200 ha, which ranged from 3.6 percent (within 3 km distance class) to 23.3 percent (15 km distance class) of all fragments. Our results suggest a road/city-effect zone of 12 to 15 km width, over which habitat loss and fragmentation extend throughout the caatinga vegetation. These findings should be considered in the regional polices for biodiversity conservation and economic development of the caatinga region

Distance from roads and cities as a predictor of habitat loss and fragmentation in the caatinga vegetation of Brazil.

Roads and cities represent huge sources of degradation for adjacent ecosystems regarding nutrient cycling, energy, water flow and species composition. In this study we test the hypothesis that distance from roads and cities is associated with habitat loss and fragmentation in the caatinga vegetation--a dry forest to scrub vegetation that covers ca. 736,000 km2 of northeast Brazil. The study site comprised a 2,828.8 km2 piece (64 km x 44.2 km) of Xingó region (09 degrees 36'S, 37 degrees 50'W), which is located between the States of Alagoas and Sergipe. Based on satellite imagery we mapped the remaining vegetation, 145 km of paved roads and the seven small-sized cities set in the study site. A positive correlation was found between the combined distance from roads and cities and the percentage of remaining vegetation as it dropped from 18% at 12 km distant to 5.9% at 1 km distant from cities and roads. Thus, remaining vegetation was reduced by one third near cities and roads. A positive correlation was also found between distance from cities and roads and the percentage of fragments larger than 200 ha, which ranged from 3.6% (within 3 km distance class) to 23.3% (15 km distance class) of all fragments. Our results suggest a road/city-effect zone of 12 to 15 km width, over which habitat loss and fragmentation extend throughout the caatinga vegetation. These findings should be considered in the regional polices for biodiversity conservation and economic development of the caatinga region.

Distance from roads and cities as a predictor of habitat loss and fragmentation in the caatinga vegetation of Brazil

Roads and cities represent huge sources of degradation for adjacent ecosystems regarding nutrient cycling, energy, water flow and species composition. In this study we test the hypothesis that distance from roads and cities is associated with habitat loss and fragmentation in the caatinga vegetation- a dry forest to scrub vegetation that covers ca. 736,000 km² of northeast Brazil. The study site comprised a 2,828.8 km² piece (64 km x 44.2 km) of Xingó region (09°36'S, 37°50'W), which is located between the States of Alagoas and Sergipe. Based on satellite imagery we mapped the remaining vegetation, 145 km of paved roads and the seven small-sized cities set in the study site. A positive correlation was found between the combined distance from roads and cities and the percentage of remaining vegetation as it dropped from 18% at 12 km distant to 5.9% at 1 km distant from cities and roads. Thus, remaining vegetation was reduced by one third near cities and roads. A positive correlation was also found between distance from cities and roads and the percentage of fragments larger than 200 ha, which ranged from 3.6% (within 3 km distance class) to 23.3% (15 km distance class) of all fragments. Our results suggest a road/city-effect zone of 12 to 15 km width, over which habitat loss and fragmentation extend throughout the caatinga vegetation. These findings should be considered in the regional polices for biodiversity conservation and economic development of the caatinga region.

Estradas e cidades representam enormes fontes de degradação dos ecossistemas adjacentes no que se refere à ciclagem de nutrientes, energia, fluxo de água e à composição de espécies. Neste estudo testa-se a hipótese de que a distância de cidades e rodovias está associada à perda e à fragmentação da caatinga, um tipo de floresta seca e scrub que cobre ca. 736.000 km² do Nordeste do Brasil. A área de estudo compreende um polígono de 2.828,8 km² (64 km x 44,2 km) na região de Xingó (09°36'S, 37°50'W), divisa dos Estados de Alagoas e Sergipe. Com base em imagens de satélite, a vegetação remanescente, 145 km de estradas pavimentadas e sete cidades foram mapeadas dentro da área de estudo. Encontrou-se correlação positiva entre a distância combinada de cidades e rodovias e o percentual de vegetação remanescente, a qual variou de 18% a 12 km de distância para 5,9% a 1 km. Desta forma, a vegetação remanescente foi reduzida em ca. 1/3 próximo a rodovias e cidades. Encontrou-se também correlação positiva entre a distância de cidades e rodovias e o percentual de fragmentos maiores que 200 ha. Os fragmentos nesta classe de tamanho representaram entre 3,6% (na classe de 3 km de distância) e 23,3% (na classe de 15 km) de todos os fragmentos. Os resultados encontrados sugerem um efeito rodovia/cidade que se prolonga entre 12 e 15 km para o interior da vegetação adjacente. Tais resultados deveriam ser considerados na definição de áreas prioritárias para a conservação da biodiversidade e na elaboração de planos de desenvolvimento econômico da região da caatinga.

The soil seed bank during Atlantic Forest regeneration in Southeast Brazil.

A survey was conducted to determine the density and species composition of viable seeds buried in four stands of a tropical montane forest at Parque Estadual Intervales, Brazil. The objective was to understand: (1) how numbers and composition of the soil seed bank change as the forest regrows, and (2) how such changes affect the species available for regeneration if forests of different ages are cut down. In each forest stand (5, 18, 27-yr-old and a mature forest), 57 soil samples were collected (0-2.5 and 2.5-5 cm deep). Viable seed density of herbaceous species ranged between 11,003 seeds. m-2 (5-yr-old vegetation) and 482 (mature forest), and between 25 (5-yr-old vegetation) and 389 seeds. m-2 (mature forest) for woody plant species in the 0-5 cm soil layer, suggesting a decrease in seed stocks in the course of forest regeneration. Seeds buried in the 0-2.5 cm soil layer represented between 56.9% and 67.4% of all viable seeds. Most of the viable seeds belonged to weeds of Asteraceae, Poaceae, Malvaceae and Solanaceae. The results provide evidence that, in forests of different ages, the soil does not store seeds of the same key ecological groups involved in the regeneration of Atlantic forest. Allochthonous seeds from remaining patches of forest, as well as their vertebrate dispersers, are needed for forest regeneration since the soil seed bank does not store large seeds of shade-tolerant species.

The soil seed bank during Atlantic Forest regeneration in Southeast Brazil

A survey was conducted to determine the density and species composition of viable seeds buried in four stands of a tropical montane forest at Parque Estadual Intervales, Brazil. The objective was to understand: (1) how numbers and composition of the soil seed bank change as the forest regrows, and (2) how such changes affect the species available for regeneration if forests of different ages are cut down. In each forest stand (5, 18, 27-yr-old and a mature forest), 57 soil samples were collected (0-2.5 and 2.5-5 cm deep). Viable seed density of herbaceous species ranged between 11,003 seeds. m-2 (5-yr-old vegetation) and 482 (mature forest), and between 25 (5-yr-old vegetation) and 389 seeds. m-2 (mature forest) for woody plant species in the 0-5 cm soil layer, suggesting a decrease in seed stocks in the course of forest regeneration. Seeds buried in the 0-2.5 cm soil layer represented between 56.9 percent and 67.4 percent of all viable seeds. Most of the viable seeds belonged to weeds of Asteraceae, Poaceae, Malvaceae and Solanaceae. The results provide evidence that, in forests of different ages, the soil does not store seeds of the same key ecological groups involved in the regeneration of Atlantic forest. Allochthonous seeds from remaining patches of forest, as well as their vertebrate dispersers, are needed for forest regeneration since the soil seed bank does not store large seeds of shade-tolerant species

Tree species impoverishment and the future flora of the Atlantic forest of northeast Brazil.

Estimates of species extinction due to human impact on tropical forests have previously been based on the relationship between species number and area. Here we use a different approach to estimate loss of tree species in the Atlantic forest of northeast Brazil. We evaluate the characteristics of plant species, their avian dispersers and the distribution of the forest remnants on the landscape to estimate that about 33.9% of tree species in this region will become extinct on a regional scale. Because northeast Brazil is the most threatened sector of South American Atlantic forest, our results highlight the need to change the current conservation paradigm for this region. Rather than focus on the creation of isolated reserves in any medium-to-large forest remnant, a bioregional planning approach is urgently required to rescue this unique biota from extinction.

Precocious puberty due to a lipid-cell tumour of the ovary.

A 4-year-old girl with a lipid cell tumour of the ovary showed isosexual precocious pseudopuberty. The endocrine activity of the tumour led to elevated plasma levels of dehydroepiandrosterone sulphate, oestradiol, testosterone and androstenedione. After tumour resection the clinical signs of abnormal hormonal stimulation disappeared within 10 months. The girl developed precocious puberty again 2 years later without any sign of relapse. Therapy with luteinizing hormone releasing hormone agonist was effective although premature activation of the hypothalamic-pituitary-gonadal axis could not clearly be demonstrated by hormonal investigations.

R339H and P453S: CYP21 mutations associated with nonclassic steroid 21-hydroxylase deficiency that are not apparent gene conversions.

Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia, an inherited disorder of cortisol biosynthesis. All mutations thus far characterized that cause this disorder appear to result from recombinations between the gene encoding the enzyme, CYP21B (CYP21), and the adjacent pseudogene, CYP21A (CYP21P). These are either deletions caused by unequal crossing-over during meiosis or apparent transfers of deleterious sequences from CYP21A to CYP21B, a phenomenon termed gene conversion. However, a small percentage of alleles do not carry such a mutation. We analyzed DNA from a patient with the mild, nonclassic form of 21-hydroxylase deficiency, who carried one allele that had no gene conversions detectable by hybridization with oligonucleotide probes. Sequence analysis revealed that this allele carried two missense mutations, R339H and P453S, neither of which has been previously observed in CYP21A or CYP21B. Each of these mutations was introduced into CYP21 cDNA which was then expressed in COS1 cells using a vaccinia virus system. Each mutation reduced the ability of the enzyme to 21-hydroxylate 17-hydroxyprogesterone to 50% of normal and the ability to metabolize progesterone to 20% of normal. Thus, each of these mutations represents a potential nonclassic 21-hydroxylase deficiency allele that is not the result of an apparent gene conversion.

Identification of molecular defects causing congenital adrenal hyperplasia by cloning and differential hybridization of polymerase chain reaction-amplified 21-hydroxylase (CYP21) genes.

Congenital adrenal hyperplasia (CAH), one of the most common autosomal recessive disorders, is caused primarily by defects in the gene encoding steroid 21-hydroxylase, CYP21B. The molecular diagnosis of CAH, important for prenatal diagnosis, carrier detection, and a better understanding of the various clinical CAH forms, is complicated by the close proximity of a highly similar pseudogene, CYP21A, containing (and probably donating, by gene conversion-like events) most of the defects underlying CAH. In this study, we describe an efficient strategy to identify molecular defects causing CAH: polymerase chain reaction-amplified CYP21 loci are cloned and hybridized to a set of oligonucleotides, allowing rapid and allele-specific identification of all known CYP21B mutations relevant to 21-hydroxylase function. Possible new mutations can be identified by subsequent nucleic acid sequencing provided they reside within the cloned CYP21B fragment (from the TATA box to the 8th of the 10 CYP21B gene exons). Using this method, the CYP21B gene mutations of a heterozygous carrier and 25 CAH patients have been identified by oligonucleotide hybridization. All disease haplotypes seem to have been generated by recombinational events involving the CYP21A pseudogene. In 5 individuals, these data were subsequently verified by nucleic acid sequencing. The procedure can be used for diagnostic applications and may facilitate identification of new CYP21B defects.

Free alpha-subunit, free beta-subunit of human chorionic gonadotropin (hCG), and intact hCG in sera of healthy individuals and testicular cancer patients.

To determine the serum concentrations of human chorionic gonadotropin (hCG), its free beta-subunit (hCG beta), and the free alpha-subunit (free alpha) common to all human glycoprotein hormones under physiological and pathological conditions, we developed monoclonal antibody-based immunoenzymometric assays. Free alpha-subunit was detected in the sera of all healthy individuals of both sexes; hCG was measurable in sera of 54% of the men, and 46% were positive for free hCG beta; in nonpregnant women, 69.5% were positive for hCG, 68.4% for the free beta-subunit. Pathological conditions, i.e., hCG-producing tumors, were studied in vitro and in vivo. In vitro, the concentrations of hCG, free hCG beta, and free alpha in tissue-culture supernates of a choriocarcinoma cell-line ("JAR") showed a parallel pattern during time-course analysis. In vivo, in long-term follow-up studies of 13 patients with testicular cancer, serum concentrations of the three analytes paralleled each other, whether the disease was in remission or not. Because of a selective increase of free hCG beta and free alpha in 27% of seminomatous tumor patients and in 13% of the nonseminomatous patients, the percentage of tumor-marker-positive sera was increased from 15% to 42% and 57% to 70%, respectively, by the additional measurement of free hCG beta and free alpha. Thus hCG, free hCG beta, and free alpha are physiologically present in a high percentage of the sera from healthy men, and the determination of free hCG beta and free alpha, although not of prognostic value, improves the diagnostic possibilities in patients with testicular cancer.