A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the "Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro" / Um resumo das espécies de répteis e anfíbios anuros das planícies costeiras arenosas brasileiras: 31 anos de esforços de amostragem do "Laboratório de Ecologia de Vertebrados da Universidade do Estado do Rio de Janeiro"

Abstract Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called "restinga" and "campo nativo" habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

Resumo Embora atualmente exista um conjunto de estudos sobre aspectos ecológicos de algumas espécies de répteis e de anfíbios que ocorrem nas planícies costeiras arenosas brasileiras (incluindo os chamados habitats de "restinga" e de "campo nativo"), há relativamente poucas informações sobre a composição de espécies geralmente associada a esses ambientes. Durante 31 anos (1988-2019) de estudos herpetológicos realizados em restingas por nossa equipe de pesquisa do Laboratório de Ecologia de Vertebrados (Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil) nós estudamos comunidades de répteis e de anfíbios e realizamos diferentes estudos com métodos semelhantes em 70 localidades de dez diferentes Estados ao longo da costa brasileira. Nossas pesquisas resultaram em registros de 87 espécies de répteis (cinco tartarugas, dois crocodilianos, seis anfisbênios, 36 lagartos e 39 serpentes) de 24 famílias, e 77 espécies de anfíbios anuros de nove famílias. Estudamos vários tópicos de história natural sobre anuros e répteis, que resultaram na publicação de alguns estudos ecológicos específicos, especialmente em relação a algumas espécies, abrangendo ecologia populacional e de comunidades, forrageamento e dieta, horário de atividade de espécies, termorregulação, reprodução, uso do microhabitat e parasitismo por ecto e endoparasitas. Nossos resultados ao longo dessas três décadas também contribuíram para a descrição de quatro novas espécies de lagartos (Ameivula nativo, Glaucomastix littoralis, G. abaetensis e G. itabaianensis). Nossos estudos constituem uma importante contribuição para o conhecimento da ecologia de répteis e de anfíbios anuros nesses ecossistemas, bem como para a conservação dos ecossistemas de restinga. A lista de espécies apresentada neste estudo, com base em nossos registros de herpetofauna das planícies costeiras arenosas, fornece para muitas localidades ao longo da costa brasileira o conhecimento necessário sobre a ocorrência de espécies, incluindo a presença de espécies endêmicas e/ ou ameaçadas de extinção, que podem ser úteis para muitas ações de conservação.

A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the "Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro".

Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called "restinga" and "campo nativo" habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

A summary of reptile and anuran amphibian species from Brazilian sandy coastal plains: 31 years of sampling efforts of the Laboratório de Ecologia de Vertebrados, Universidade do Estado do Rio de Janeiro

Abstract Although currently there is already a set of studies regarding ecological aspects of some particular reptile and amphibian species living in Brazilian sandy coastal plains (including the so-called restinga and campo nativo habitats), there is comparatively few information on the species composition usually associated to these environments. During 31 years (1988-2019) of herpetological studies carried out in sandy coastal plains environments by our research team of the Laboratory of Vertebrate Ecology (Department of Ecology, Universidade do Estado do Rio de Janeiro, in Rio de Janeiro Brazil) we have surveyed reptile and amphibian communities and performed different studies with similar methods in 70 sites from 10 different states along the Brazilian coast. Our surveys resulted in records of 87 species of reptile (five turtles, two crocodylians, six amphisbaenians, 36 lizards and 39 snakes) from 24 families, and 77 species of anuran amphibians from nine families. We have studied multiple natural history topics for anurans and reptiles which resulted in the publication of some specific ecological studies, especially regarding some species, encompassing population and community ecology, foraging and feeding habits, species activity, thermoregulation, reproduction, use of microhabitats, and parasitism by ecto and endoparasites. Our results along these three decades have also contributed for the description of four new lizard species (Ameivula nativo, Glaucomastix littoralis, G. abaetensis and G. itabaianensis). Our studies constitute an important contribution to the knowledge of the ecology of anuran amphibians and reptiles in these ecosystems, as well as to the conservation of sandy coastal plains environment. The checklist presented in this study, based on our records of sandy coastal plains herpetofauna, provides for many localities along the Brazilian coast, the needed knowledge on species occurrence, including the presence of endemic and/or endangered species, which can be of value for many conservation actions.

Resumo Embora atualmente exista um conjunto de estudos sobre aspectos ecológicos de algumas espécies de répteis e de anfíbios que ocorrem nas planícies costeiras arenosas brasileiras (incluindo os chamados habitats de restinga e de campo nativo), há relativamente poucas informações sobre a composição de espécies geralmente associada a esses ambientes. Durante 31 anos (1988-2019) de estudos herpetológicos realizados em restingas por nossa equipe de pesquisa do Laboratório de Ecologia de Vertebrados (Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil) nós estudamos comunidades de répteis e de anfíbios e realizamos diferentes estudos com métodos semelhantes em 70 localidades de dez diferentes Estados ao longo da costa brasileira. Nossas pesquisas resultaram em registros de 87 espécies de répteis (cinco tartarugas, dois crocodilianos, seis anfisbênios, 36 lagartos e 39 serpentes) de 24 famílias, e 77 espécies de anfíbios anuros de nove famílias. Estudamos vários tópicos de história natural sobre anuros e répteis, que resultaram na publicação de alguns estudos ecológicos específicos, especialmente em relação a algumas espécies, abrangendo ecologia populacional e de comunidades, forrageamento e dieta, horário de atividade de espécies, termorregulação, reprodução, uso do microhabitat e parasitismo por ecto e endoparasitas. Nossos resultados ao longo dessas três décadas também contribuíram para a descrição de quatro novas espécies de lagartos (Ameivula nativo, Glaucomastix littoralis, G. abaetensis e G. itabaianensis). Nossos estudos constituem uma importante contribuição para o conhecimento da ecologia de répteis e de anfíbios anuros nesses ecossistemas, bem como para a conservação dos ecossistemas de restinga. A lista de espécies apresentada neste estudo, com base em nossos registros de herpetofauna das planícies costeiras arenosas, fornece para muitas localidades ao longo da costa brasileira o conhecimento necessário sobre a ocorrência de espécies, incluindo a presença de espécies endêmicas e/ ou ameaçadas de extinção, que podem ser úteis para muitas ações de conservação.

Differential success in sampling of Atlantic Forest amphibians among different periods of the day.

In general, anurans tend to be nocturnal, though diurnal activity is characteristic of some groups. Studies show that frog activity may be inferred based on the number of individuals collected at different periods of the day, during large-scale field surveys. We investigated the best period of the day to conduct amphibian sampling in nine Atlantic Rainforest areas in southeastern Brazil, based on intensive field surveys. At each locality we employed similar sampling effort during diurnal, crepuscular and nocturnal searches (totaling 704.5 sampling hours). We pooled data from all localities for each period and estimated the proportion of frogs of each species active at each period based on the total number of individuals and on the number of species found during all surveys for that period. We recorded a total of 817 individual frogs from 69 species. Species richness was highest at night (median = 12 species), intermediate at dusk (median = 8), and lowest during the day (median = 4). The percentage of the total number of individual frogs found (pooled species) was highest during the night (ca. 53%) and lowest during the day (ca. 14%). Analyzing each species separately, the number of individuals recorded was consistently higher at dusk and night for most species. Our study evidences a trend for nocturnal activity for most Atlantic Rainforest frogs, with few species having primarily diurnal habits. Those results may favor future studies and conservation efforts for amphibian species.

Species composition, richness and nestedness of lizard assemblages from Restinga habitats along the brazilian coast.

Habitat fragmentation is well known to adversely affect species living in the remaining, relatively isolated, habitat patches, especially for those having small range size and low density. This negative effect has been critical in coastal resting habitats. We analysed the lizard composition and richness of restinga habitats in 16 restinga habitats encompassing three Brazilian states (Rio de Janeiro, Espírito Santo and Bahia) and more than 1500km of the Brazilian coast in order to evaluate if the loss of lizard species following habitat reduction occur in a nested pattern or at random, using the "Nestedness Temperature Calculator" to analyse the distribution pattern of lizard species among the restingas studied. We also estimated the potential capacity that each restinga has to maintain lizard species. Eleven lizard species were recorded in the restingas, although not all species occurred in all areas. The restinga with the richest lizard fauna was Guriri (eight species) whereas the restinga with the lowest richness was Praia do Sul (located at Ilha Grande, a large coastal island). Among the restingas analysed, Jurubatiba, Guriri, Maricá and Praia das Neves, were the most hospitable for lizards. The matrix community temperature of the lizard assemblages was 20.49° (= P <0.00001; 5000 randomisations; randomisation temperature = 51.45° ± 7.18° SD), indicating that lizard assemblages in the coastal restingas exhibited a considerable nested structure. The degree in which an area is hospitable for different assemblages could be used to suggest those with greater value of conservation. We concluded that lizard assemblages in coastal restingas occur at a considerable level of ordination in restinga habitats and that some restinga areas such as Jurubatiba, Guriri, Maricá and Praia das Neves are quite important to preserve lizard diversity of restinga environments.

[About the functional aspect of septorhinoplasty]. / Der Funktionelle Aspekt der Septorhinoplastik.

INTRODUCTION: Rhinosurgeons emphasize the functional aspect of septorhinoplasty (SRP). So far only a few publications with exact data exist. PATIENTS AND METHODS: Fifty-two patients (33 male symbol; 19 female symbol, average age: 37 years) were examined before and approx. 1 year after SRP. Rhinomanometry, olfactometry, and photographic documentation were carried out. Using a questionnaire they were asked about the functional outcome and about their original personal aims concerning the SRP. RESULTS: All patients suffered from a deviated septum. Regarding the external aspect 38 patients had a crooked nose with or without a hump, 7 patients had a hump without external deviated nose, and 7 patients had a saddle nose or other deformities. If total nasal airflow is considered rhinomanometry showed no significant improvement postoperatively (p=0.115). If only the worse nasal side is considered rhinometry showed a highly significant improvement (p=0.002). In the questionnaire 34 patients (65%) indicated an improvement, 14 patients (27%) no change, and 4 patients a deterioration of the nasal airflow. The final aesthetic outcome was assessed by 39 patients (75%) as an improvement, by 12 patients (23%) as unchanged, and by 1 patient (2%) as worse. There were no significant changes in the olfactory function. Asked about their personal aims 29 patients (56%) wished first of all an improvement of nasal airflow, 5 patients (10%) a good aesthetic result, and 18 patients (34%) both aspects. CONCLUSIONS: The results show that use of the term "functional" SRP is justified. In the majority of cases a simultaneously existing functional-plastic problem is solved. Our results support the importance of rhinomanometry as an objective parameter in the perioperative diagnostic battery of nasal obstruction.

A central domain binding site in insulin-like growth factor binding protein-5 for the acid-labile subunit.

Like insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-5 forms a ternary complex with insulin-like growth factor (IGF)-I or IGF-II, and the acid-labile subunit (ALS). The study of IGFBP-5/IGFBP-6 chimeric proteins with amino-terminal and middle domain swaps, has revealed the existence of a site in the middle domain of IGFBP-5, that binds to ALS in the absence of the IGFBP-5 carboxy-terminal domain. An IGFBP-6 chimeric protein containing the central domain of IGFBP-5 complexed efficiently with ALS, and a carboxy-terminally truncated IGFBP-5 mutant, IGFBP-5'(1-169), also bound to ALS in the presence of IGFs, although with much less potency than full length rhIGFBP-5. In contrast to the latter, IGFBP-5(1-169) preferentially formed ternary complexes with IGF-II rather than IGF-I. These results indicate that a site which binds ALS exists in IGFBP-5 mutants which lack the IGFBP-5 carboxy-terminal domain.

Analysis of the Bak protein expression in human polymorphonuclear neutrophils.

In this study, we investigated the expression of Bak, a member of the Bcl-2 protein family, in human polymorphonuclear neutrophils. Northern blot and Western blot analyses revealed that Bak messenger RNA and protein were constitutively expressed in peripheral polymorphonuclear neutrophils and mononuclear cells, as well as in several hematopoietic cell lines. Remarkably, culturing neutrophils for 24 h in the presence or absence of interferon-gamma or tumor necrosis factor-alpha, which have been described to modulate the survival rate of these cells, did not influence the expression of antigenic Bak. Taken together, our data indicate that the expression of the pro-apoptotic protein Bak in polymorphonuclear neutrophils is constitutive, is not subject to modulation, and does not correlate with the neutrophil life span in culture.

Insulin-like growth factor-binding protein 5 complexes with the acid-labile subunit. Role of the carboxyl-terminal domain.

We have recently shown that insulin-like growth factor (IGF)-binding protein 5 forms ternary complexes with IGF-I or IGF-II and the acid-labile subunit (ALS) (Twigg, S. M., and Baxter, R. C. (1998) J. Biol. Chem. 273, 6074-6079). Because IGF-binding protein 3 (IGFBP-3) binds to ALS through its basic carboxyl-terminal domain, we tested whether a homologous region present in IGFBP-5 is involved in IGFBP-5 binding to ALS. Chimeric peptides were generated by carboxyl-terminal domain interchange between recombinant human IGF-BP-5 and IGFBP-6, producing two IGFBP peptides designated 5-5-6 and 6-6-5. Determined by immunoprecipitation and by Superose chromatography, 6-6-5 formed ternary complexes, albeit less potently than IGF-BP-5. In contrast, 5-5-6, like IGFBP-6, did not form ternary complexes by these methods. Whereas 6-6-5, like IGFBP-6, had a marked preference for binary complex formation with IGF-II rather than IGF-I, it formed ternary complexes more efficiently with IGF-I, like IGF-BP-5. The glycosaminoglycans heparin and heparan sulfate bind to IGFBP-5 through its basic carboxyl-terminal domain. At high concentrations, these glycosaminoglycans inhibited ALS binding to binary complexed IGF-BP-5. In addition, in the absence of IGFs, IGFBP-5, a synthetic peptide representing the basic carboxyl-terminal sequence IGFBP-5(201-218), and the corresponding IGFBP-3 basic sequence IGFBP-3(215-232), competed weakly for ALS binding to covalent IGF-IGFBP-5 complex, as did a random-sequence synthetic peptide with the same composition as IGFBP-5(201-218). These findings are consistent with the basic carboxyl-terminal domain on IGFBP-5 being the principal site in IGFBP-5 that binds to ALS.

Genomic structure and domain organisation of the human Bak gene.

The Bcl-2 homologue, Bak, is a potent inducer of apoptosis. FISH data presented here located the gene to 6p21.3. Mapping was consistent with its location centromeric of the HSET locus and approximately 400kb from the MHC. The construction of a contig of genomic clones across the locus facilitated the sequencing of a PAC containing the gene. Comparison of the gene structure to functional and physical domains revealed a good agreement between the physical structure and the intron-exon organisation. The position of a single intron was conserved in comparison to other members of the Bcl-2 family, namely Bax, CED-9, Bcl-X and Bcl-2, but all other introns were displaced, consistent with a divergent phylogeny.

Edg-2/Vzg-1 couples to the yeast pheromone response pathway selectively in response to lysophosphatidic acid.

We have functionally expressed the human cDNA encoding the putative lysophosphatidic acid (LPA) receptor Edg-2 (Vzg-1) in Saccharomyces cerevisiae in an attempt to determine the agonist specificity of this G-protein-coupled receptor. LPA activated the pheromone response pathway in S. cerevisiae expressing Edg-2 in a time- and dose-dependent manner as determined by induction of a pheromone-responsive FUS1::lacZ reporter gene. LPA-mediated activation of the pheromone response pathway was dependent on mutational inactivation of the SST2 gene, the GTPase-activating protein for the yeast G alpha protein (the GPA1 gene product). This indicates that, in sst2 delta yeast cells, Edg-2 can efficiently couple to the yeast heterotrimeric G-protein in response to LPA and activate the yeast mitogen-activated protein kinase pathway. The Edg-2 receptor showed a high degree of specificity for LPA; other lyso-glycerophospholipids, sphingosine 1-phosphate, and diacyl-glycerophospholipids did not activate FUS1::lacZ. LPA analogs including a cyclic phosphoester form and ether-linked forms of LPA activated FUS1::lacZ, although fatty acid chains of 6 and 10 carbons did not activate FUS1::lacZ, suggesting a role for the side chain in ligand binding or receptor activation. These results indicate that Edg-2 encodes a highly specific LPA receptor.

Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression.

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Fas-mediated apoptosis in human prostatic carcinoma cell lines.

Of six prostatic carcinoma cell lines examined (ALVA31, DU145, JCA1, LNCaP, ND1, and PC3) by flow cytometric analysis, all were found to be positive for Fas antigen. Furthermore, of the prostate tissue specimens studied (six cases), all revealed Fas expression in benign and malignant epithelial cells. The agonistic anti-Fas monoclonal antibody (IPO-4) induced apoptosis in only two of six cell lines investigated, PC3 and ALVA31. PCR analysis indicated that all cell lines expressed normal transmembrane and death domains of Fas antigen. Using Western blot analysis, we found abundant expression of p53 in the cytoplasm of two Fas-resistant cell lines, DU145 and ND1, and did not find p53 in two Fas-sensitive cell lines, PC3 and ALVA31. Western blot and PCR analysis did not show consistent differences between cell lines examined in the expression of Bcl-2, Bcl-X(L), Bcl-X(S), and Bak. In contrast, Bax protein was not detected in two Fas-resistant cell lines, DU145 and ND1. We also showed that three Fas-resistant cell lines, DU145, ND1, and JCA1, expressed CD40, whereas the two Fas-sensitive cell lines, PC3 and ALVA31, were CD40 negative. Fas-sensitive cell lines were transfected with the cDNA encoding CD40, and the CD40-positive transfectant became more resistant to growth inhibition mediated by treatment with TNF-alpha and anti-Fas monoclonal antibody. Treatment with cycloheximide converted the phenotype of resistant cell lines from Fas resistant to Fas sensitive. Moreover, anti-Fas treatment of both resistant and sensitive cell lines induced rapid tyrosine phosphorylation or dephosphorylation of multiple proteins. These results suggest that the apoptotic machinery involved in DNA fragmentation is already in place in Fas-resistant cell lines, and thus, Fas-mediated apoptosis could be a target for therapeutic intervention.

Molecular cloning and characterization of Porphyromonas gingivalis lysine-specific gingipain. A new member of an emerging family of pathogenic bacterial cysteine proteinases.

The proteinases of Porphyromonas gingivalis are key virulence factors in the etiology and progression of periodontal disease. Previous work in our laboratories resulted in the purification of arginine- and lysine-specific cysteine proteinases, designated gingipains, that consist of several tightly associated protein subunits. Recent characterization of arginine-specific gingipain-1 (gingipain R1; RGP-1) revealed that the sequence is unique and that the protein subunits are initially translated as a polyprotein encoding a proteinase domain and multiple adhesin domains (Pavloff, N., Potempa, J., Pike, R. N., Prochazka, V., Kiefer, M. C., Travis, J., and Barr, P. J. (1995) J. Biol. Chem. 270, 1007-1010). We now show that the lysine-specific gingipain (gingipain K; KGP) is also biosynthesized as a polyprotein precursor that contains a proteinase domain that is 22% homologous to the proteinase domain of RGP-1 and multiple adhesin domains. This precursor is similarly processed at distinct sites to yield active KGP. The key catalytic residues in the proteinase domain of KGP are identical to those found in RGP-1, but there are significant differences elsewhere within this domain that likely contribute to the altered substrate specificity of KGP. Independent expression of the proteinase domain in insect cells has shown that KGP does not require the presence of the adhesin domains for correct folding to confer proteolytic activity.

SARPs: a family of secreted apoptosis-related proteins.

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.

Maspin is an intracellular serpin that partitions into secretory vesicles and is present at the cell surface.

The tumor suppressor maspin (mammary serpin) was originally identified as a component of human mammary epithelial cells that is downregulated as mammary tumor cells progress from the benign to the invasive and metastatic states. Maspin inhibits cellular invasion, motility, and proliferation, but its mechanism of action is currently unknown. Because the cellular machinery responsible for these processes is cytoplasmic, we have reexamined the tissue distribution and subcellular localization of maspin. We find that maspin, or a maspin-like protein, is present in many human organs, in which it localizes to epithelia. In cultured human mammary myoepithelial cells, maspin is predominantly a soluble cytoplasmic protein that associates with secretory vesicles and is present at the cell surface. In vitro assays show that the vesicle association is due to the existence of an uncleaved facultative secretion signal that allows small amounts of maspin to partition into the endoplasmic reticulum. These results demonstrate that maspin is more widespread than previously believed. The subcellular localization studies indicate that soluble intracellular and vesicle-associated maspin probably play an important role in controlling the invasion, motility, and proliferation of cells expressing it, whereas extracellular maspin may also regulate these processes in adjacent cells.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes.

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

Identification and cloning of a locus of serine repeat antigen (sera)-related genes from Plasmodium vivax.

Polymerase chain reaction (PCR) primers based on the cysteine proteinase-like active site regions of the Plasmodium falciparum serine repeat antigen (SERA) were used to identify related sequences within the genome of P. vivax. Molecular cloning and sequence analysis of approximately 25 kb of P. vivax genomic DNA revealed a cluster of five repeated SERA-like genes (V-SERA-1-5), each encoding a cysteine proteinase-related protein. In addition to DNA sequence homology, significant similarities in deduced intron/exon organizations were also observed. The characteristic polyserine sequence found in SERA was not present in any of the deduced V-SERA sequences. Instead, in this region of the five genes, considerable sequence differences were found, suggesting the potential for antigenic variation in the V-SERA molecules. In common with SERA, however, the codon at the position corresponding to the active site cysteine residue of active mammalian and plant cysteinyl proteinases was found to be that of a serine residue in each of the V-SERA genes. Furthermore, in four of the five genes, including the expressed V-SERA-5 gene, the codon for the active site histidine residue was changed to that of a leucine residue. These critical differences reinforce the concept that a biological activity other than proteolysis is likely to be the primary function of the proteins encoded by this family of genes.

The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. Evidence that maspin is not a protease inhibitory serpin.

The role of tumor suppressor proteins in the development of malignancy has made the understanding of their molecular mechanisms of action of great importance. Maspin is a tumor suppressor produced by a number of cell types of epithelial origin. Exogenous recombinant maspin has been shown to block the growth, motility, and invasiveness of breast tumor cell lines in vitro and in vivo. Although belonging to the the serine proteinase inhibitor (serpin) superfamily of proteins, the molecular mechanism of maspin is currently unknown. Here we show that the reactive site loop of maspin exists in an exposed conformation that does not require activation by cofactors. The reactive site loop of maspin, however, does not act as an inhibitor of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and trypsin but rather as a substrate. Maspin is also unable to inhibit tissue and urokinase type plasminogen activators. Stability studies show that maspin cannot undergo the stressed-relaxed transition typical of proteinase-inhibitory serpins, and the protein is capable of spontaneous polymerization induced by changes in pH. It is likely, therefore, that maspin is structurally more closely related to ovalbumin and angiotensinogen, and its tumor suppressor activity is independent of a latent or intrinsic trypsin-like serine proteinase-inhibitory activity.

Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak.

Members of the Bcl-2 family of proteins are characterized by their ability to modulate cell death. Bcl-2 and some of its homologues inhibit apoptosis, whereas other family members, such as Bax, will accelerate apoptosis under certain conditions. Here we describe the identification and characterization of a complementary DNA that encodes a previously unknown Bcl-2 homologue designated Bak. Like Bax, the bak gene product primarily enhances apoptotic cell death following an appropriate stimulus. Unlike Bax, however, Bak can inhibit cell death in an Epstein-Barr-virus-transformed cell line. The widespread tissue distribution of Bak messenger RNA, including those containing long-lived, terminally differentiated cell types, suggests that cell-death-inducing activity is broadly distributed, and that tissue-specific modulation of apoptosis is controlled primarily by regulation of molecules that inhibit apoptosis.