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PubMed1996

Cloning, expression and genomic organization of human placental protein disulfide isomerase (previously identified as phospholipase C alpha).

Phosphoinositol-specific Phospholipase C plays an important role in transducing receptor generated signals to the rest of the cell. A cDNA encoding a phospholipase has been described (Bennett et al., 1988, Nature 334, 268-270). However it is probable that this cDNA in fact encodes a protein disulfide isomerase. Since the original work suggested that this enzyme was important in the reproductive tract we sort to clone, sequence, express and characterize the recombinant protein isolated from the placenta. We have cloned and sequenced the cDNA encoding the human homolog of this cDNA from human placenta, although the mRNA was widespread in the female reproductive tract. We have transiently expressed it in both COS cells and also 1BR fibroblasts. Cell lysates were assayed for increased phospholipase activity and protein disulfide activity. We describe the entire cDNA sequence which is highly conserved between species. We have also cloned a portion of the genomic gene and described the intron/exon boundaries. In vitro translation of this cDNA showed that it encoded a protein of 61 kD with a cleavable signal peptide. Transient expression showed the protein produced had no phospholipase activity but did show protein disulfide isomerase activity. The expression work shows that this cDNA indeed encodes a protein disulfide isomerase and not a phospholipase. The nucleotide sequence shows marked conservation of the coding and regulatory regions which may suggest that this enzyme has evolved to perform a highly specialized function.

Base Sequence↗
PubMed1997

A neurohormone regulating both methyl farnesoate synthesis and glucose metabolism in a crustacean.

Methyl farnesoate (MF) has been identified as a juvenile hormone-like compound in crustacea which has central roles in the regulation of development and reproduction. To study the regulation of MF synthesis, we isolated a neuropeptide which inhibits MF synthesis from the neurohemal organ-sinus gland X-organ complex of the spider crab Libinia emarginata. The primary structure of this neuropeptide has been determined. It has 72 amino acid residues (deduced molecular mass 8490.5 Da) with pyroglutamic acid at the N-terminus and NH3 at the C-terminus. It shares a high percentage of sequence identity with other sinus gland neuropeptids which form the unique family of CHH neuropeptides of crustacea. Activity studies showed that this neurohormone has dual effects: it inhibited MF synthesis in vitro and had hyperglycemic activity when injected into crabs.

Amino Acid Sequence↗
PubMed1995

A salinity-induced gene from the halophyte M. crystallinum encodes a glycolytic enzyme, cofactor-independent phosphoglyceromutase.

In the facultative halophyte Mesembryanthemum crystallinum (ice plant), salinity stress triggers significant changes in gene expression, including increased expression of mRNAs encoding enzymes involved with osmotic adaptation to water stress and the crassulacean acid metabolism (CAM) photosynthetic pathway. To investigate adaptive stress responses in the ice plant at the molecular level, we generated a subtracted cDNA library from stressed plants and identified mRNAs that increase in expression upon salt stress. One full-length cDNA clone was found to encode cofactor-independent phosphoglyceromutase (PGM), an enzyme involved in glycolysis and gluconeogenesis. Pgm1 expression increased in leaves of plants exposed to either saline or drought conditions, whereas levels of the mRNA remained unchanged in roots of hydroponically grown plants. Pgm1 mRNA was also induced in response to treatment with either abscisic acid or cytokinin. Transcription run-on experiments confirmed that Pgm1 mRNA accumulation in leaves was due primarily to increased transcription rates. Immunoblot analysis indicated that Pgm1 mRNA accumulation was accompanied by a modest but reproductible increase in the level of PGM protein. The isolation of a salinity-induced gene encoding a basic enzyme of glycolysis and gluconeogenesis indicates that adaptation to salt stress in the ice plant involves adjustments in fundamental pathways of carbon metabolism and that these adjustments are controlled at the level of gene expression. We propose that the leaf-specific expression of Pgm1 contributes to the maintenance of efficient carbon flux through glycolysis/gluconeogenesis in conjunction with the stress-induced shift to CAM photosynthesis.

Adaptation, Biological↗
PubMed1995

Evolutionary aspects of gonadotropin-releasing hormone and its receptor.

1. Gonadotropin-releasing hormone (GnRH) was originally isolated as a hypothalamic peptide hormone that regulates the reproductive system by stimulating the release of gonadotropins from the anterior pituitary. However, during evolution the peptide was subject to gene duplication and structural changes, and multiple molecular forms have evolved. 2. Eight variants of GnRH are known, and at least two different forms are expressed in species from all vertebrate classes: chicken GnRH II and a second, unique, GnRH isoform. 3. The peptide has been recruited during evolution for diverse regulatory functions: as a neurotransmitter in the central and sympathetic nervous systems, as a paracrine regulator in the gonads and placenta, and as an autocrine regulator in tumor cells. 4. Evidence suggests that in most species the early-evolved and highly conserved chicken GnRH II has a neurotransmitter function, while the second form, which varies across classes, has a physiologic role in regulating gonadotropin release. 5. We review here evolutionary aspects of the family of GnRH peptides and their receptors.

Amino Acid Sequence↗
PubMed1995

Structural analysis of the carbohydrate moiety of arabinogalactan-proteins from stigmas and styles of Nicotiana alata.

Arabinogalactan-proteins (AGPs) from the female reproductive tissues (stigmas and styles) of Nicotiana alata were isolated from the saturated ammonium sulfate supernatant of buffer-soluble extracts by precipitation with the beta-glucosyl Yariv reagent, followed by gel-filtration chromatography under dissociating conditions. The AGPs had characteristics typical of other AGPs: a high proportion of carbohydrate (95%) with a high ratio of Gal p to Ara f (2:1), and a low protein content (5%) with high levels of alanine, serine, and hydroxyproline. The AGPs consisted of a major species which was almost neutral, and a minor species which was more negatively charged. Sedimentation equilibrium experiments showed that the purified AGPs had a weight-average molecular weight of 143 kD. Linkage analysis showed that the AGPs contained a highly branched backbone of 3-, 6-, and 3,6-linked Gal p residues, bearing terminal Gal p and terminal Ara f residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR spectroscopy confirmed the presence of these glycosyl linkage types, and showed a high mobility of the terminal Ara f residues consistent with their location on the periphery of the molecules. This analysis represents the most complete 1H assignment for AGP molecules in solution. No difference in the carbohydrate analyses was found between AGPs isolated separately from stigmatic or stylar tissue, or between AGPs isolated from stigmas and styles of plants of different self-incompatibility genotypes.

Arabinose↗
PubMed1989

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa.

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.

Ammonium Sulfate↗
PubMed1989

The relation between oral movement control and speech.

A large series of neurological patients, selected solely on the basis that they had damage restricted to one hemisphere of the brain, was given a variety of tests of basic speech and praxic function. Within the left-damaged group, patients were further identified as aphasic or nonaphasic, based on preexisting standard tests of aphasia. Subgroups of aphasics were studied on the basis of lesion location, rather than on the basis of aphasia type. The focus of the study was the relation between the production of speech and nonspeech oral movements, particularly across anterior and posterior lesions. Reproduction of single nonverbal oral movements and of single isolated speech sounds was found to be very highly correlated, and both depended selectively on the left anterior region of the brain. This same region was critically important for rapid repeated articulation of a syllable, suggesting that it mediates control at some "unit" level of movement, in a phenomenological sense, for both speech and nonspeech movements. Other "speech" regions in the left hemisphere appeared to be dispensable for the production of single oral movements, whether these were verbal or nonverbal movements. However, for most aphasic patients, an area in the left posterior region was inferred to be essential for production of multiple oral movements, whether nonverbal or verbal, suggesting a critical role in the accurate selection of movements. Within the posterior region, there was further differentiation for multisyllabic speech into a parietal system, which appeared to mediate primarily praxic function, and a temporal system, which appeared to mediate verbal-echolalic function. Aphasias from anterior and posterior lesions resembled "Broca's" and "Wernicke's" aphasia only insofar as they differed in fluency, with anterior aphasics clearly less fluent. Tests of speech comprehension did not differentiate the groups. It is suggested that classifying aphasic patients via lesion location rather than aphasic typology might yield a view of functional subsystems different from those commonly accepted.

Adult↗
PubMed1989

Comment on the status of Echinococcus granulosus in the UK.

Echinococcus granulosus is composed of a complex of strains, the status of which has caused controversy and doubt. All have different characteristics which have arisen from animal husbandry practices, resulting in isolation and restriction of gene flow, and/or from the reproductive characteristics of the organisms. Whatever the mechanism for strain variation, Don McManus, Alan Lymbery and Andrew Thompson have no doubts that distinct variants occur in spite of a recent publication that suggests there is only one form of E. granulosus in the UK.

Journal Article↗
PubMed1988

Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae.

Vegetative and reproductive stages of Caecomyces equi gen. nov., sp. nov. isolated from the horse caecum were examined by light and electron microscopy. This organism, which is similar to isolates known as Sphaeromonas communis, produces uniflagellate, uninucleate zoospores whose perikinetosomal structures, i.e. circumflagellar ring, spur, struts and scoop, are similar in many respects to those described in species of Neocallimastix. Microtubular roots extend basally from the spur and associate with hydrogenosomes and the nucleus. Another group of microtubules radiates laterally in a fan-shaped array close to the plasmalemma. Zoospores encyst, shedding their flagella with basal bodies, and germinate to diglobular thalli. Either coralloid or bulbous rhizoids form in plant material, but only the latter in axenic culture. Incipient zoospores are produced from a multinucleate eucarpic thallus and devlop within cleavage vacuoles containing flagella. An isolate from the cow rumen was found to be similar to C. equi in morphology and zoospore ultrastructure. On the basis of zoospore ultrastructure, we assign the new genus to the Neocallimasticaceae of the order Spizellomycetales. Organisms previously described as Sphaeromonas communis and Piromonas communis are renamed Caecomyces communis and Piromyces communis and assigned to the same family.

Animals↗
PubMed2004

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae).

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.

Animals↗
PubMed2004

Separation of human immunodeficiency virus type 1 from motile sperm by the double tube gradient method versus other methods.

OBJECTIVE: To compare a new sperm-processing device, utilizing a double tube and gradient, with other commonly used sperm-processing methods for their abilities to exclude human immunodeficiency virus type 1 (HIV-1) from the motile sperm fraction while retaining maximum sperm yield. DESIGN: Laboratory experiments. SETTING: Academic research environment. PATIENT(S): Healthy HIV-1 seronegative men between the ages of 25 and 55. INTERVENTION(S): Semen samples were spiked with HIV-1 (MN HIV-1 strain; range of concentrations: 10(-1)-10(6) TCID(50)) and subjected to one of the following sperm-processing methods: double sperm tube with discontinuous gradient of sperm separation medium formed inside (double tube gradient), conventional single tube gradient, swim-up or single tube gradient followed by swim-up (gradient/swim-up), which is the method currently used to minimize HIV-1 in semen used for assisted reproductive technology (ART) procedures. For the gradient techniques, Percoll, ISolate, and PureSperm sperm separation media were compared for efficacy of HIV-1 removal. The amount of HIV-1 remaining in the motile sperm pellet after processing was measured by reverse transcription-polymerase chain reaction (RT-PCR), NucliSens assay, or quantitative HIV-1 culture. A hemacytometer was used to microscopically assess motile sperm count. MAIN OUTCOME MEASURE(S): HIV-1 RNA copy number, decrease in HIV-1 TCID(50), motile sperm yield. RESULT(S): The double tube gradient technique was significantly better than all other methods in the removal of HIV-1 from the motile sperm fraction, and produced significantly higher sperm yields in comparison with the gradient/swim-up method. CONCLUSION(S): The double tube gradient technique is a relatively simple and effective method that reduces the risk of infection by HIV-1 while producing good sperm recovery.

Adult↗
PubMed2006

Full genome sequence and some biological properties of reticuloendotheliosis virus strain APC-566 isolated from endangered Attwater's prairie chickens.

Reticuloendotheliosis virus (REV) causes runting, high mortality, immunosuppression, and chronic neoplasia associated with T and/or B cell lymphomas in a variety of domestic and wild birds, including Attwater's prairie chickens (APC) (Tympanuchus cupido attwateri). The complete proviral sequence of a recent REV isolate from APC (REV APC-566) was determined. This virus was isolated from an APC maintained in captivity in a reproduction program intended to avoid its extinction. REV APC-566 was determined to be oncogenic in Japanese quail (Coturnix coturnix japonica), chickens (Gallus gallus) and turkeys (Meleagris gallopavo). Immune responses against bacteria and viruses were significantly reduced in turkeys infected with REV APC-566. The proviral genome is 8286 nucleotides in length and exhibits a genetic organization characteristic of replication-competent gammaretroviruses. The REV APC-566 provirus contains two identical long terminal repeats (LTR) and a complete set of genes including gag, gag-pol and env. As previously reported, alignments with other REV sequences showed high similarity with sequences found in the gag and pol genes from other REVs. The REV APC-566 env gene showed high nucleotide sequence homology with REV sequences inserted in fowl poxvirus (99.8%), and with spleen necrosis virus (SNV) (95.1%). Sequences coding for a previously reported immunosuppressive peptide contained in the transmembrane region of the env gene are well conserved among all REV sequences analyzed. The LTR was the most divergent region, exhibiting various deletions and insertions. REV APC-566 has a unique insertion of 23 bp in U3 and shares deletions of 19 and 5 bp with chicken syncytial virus and REV inserts in fowlpox virus.

Amino Acid Sequence↗
PubMed1999

Mice deficient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction.

The lats gene has been identified as a tumour suppressor in Drosophila melanogaster using mosaic screens. Mosaic flies carrying somatic cells that are mutant for lats develop large tumours in many organs. The human LATS1 homologue rescues embryonic lethality and inhibits tumour growth in lats mutant flies, demonstrating the functional conservation of this gene. Biochemical and genetic analyses have revealed that LATS1 functions as a negative regulator of CDC2 (ref. 3). These data suggest that mammalian LATS1 may have a role in tumorigenesis. To elucidate the function of mammalian LATS1, we have generated Lats1-/- mice. Lats1-/- animals exhibit a lack of mammary gland development, infertility and growth retardation. Accompanying these defects are hyperplastic changes in the pituitary and decreased serum hormone levels. The reproductive hormone defects of Lats1-/- mice are reminiscent of isolated LH-hypogonadotropic hypogonadism and corpus luteum insufficiency in humans. Furthermore, Lats1-/- mice develop soft-tissue sarcomas and ovarian stromal cell tumours and are highly sensitive to carcinogenic treatments. Our data demonstrate a role for Lats1 in mammalian tumorigenesis and specific endocrine dysfunction.

Animals↗
PubMed2002

Convergent development of low-relatedness supercolonies in Myrmica ants.

Many ant species have independently evolved colony structures with multiple queens and very low relatedness among nestmate workers, but it has remained unclear whether low-relatedness kin structures can repeatedly arise in populations of the same species. Here we report a study of Danish island populations of the red ant Myrmica sulcinodis and show that it is likely that such repeated developments occur. Two microsatellite loci were used to estimate genetic differentiation (F(ST)) among three populations and nestmate relatedness within these populations. The F(ST) values were highly significant due to very different allele frequencies among the three populations with relatively few common alleles and relatively many rare alleles, possibly caused by single queen foundation and rare subsequent immigration. Given the isolation of the islands and the low investment in reproduction, we infer that each of the populations was most likely established by a single queen, even though all three extant populations now have within-colony relatedness 95%), and the genetic differentiation of nests showed a significantly positive correlation with the distance between them. Both male-biased sex-ratio and genetic viscosity are expected characteristics of populations where queens have very local dispersal and where new colonies are initiated through nest-budding. Based on a comparison with other M. sulcinodis populations we hypothesise a distinct succession of population types and suggest that this may be a possible pathway to unicoloniality, ie, development towards a complete lack of colony kin structure and unrelated nestmate workers.

Animals↗
PubMed2001

Haplotype variation of cpDNA in the agamic grass complex Pennisetum section Brevivalvula (Poaceae).

The Brevivalvula section of the grass polyploid complex Pennisetum shows various reproductive systems, apomixis being the most widespread. Haplotype variation of chloroplast DNA was studied in the six morphological taxa (species) of this section by using RFLP analysis in 54 plants corresponding to 14 elementary taxa, each characterized on the basis of morphology and ploidy level. Two additional species, Pennisetum glaucum and P. purpureum, which belong to another section of the same genus, were analysed for comparison. In Brevivalvula, chloroplast DNA size was estimated to range between 130 and 133 kb. Thirteen of 15 distinct haplotypes identified in the study were specific to the Brevivalvula section. They were unequally distributed among the morphotypes, the ploidy levels and sampling sites. Within the Brevivalvula section, plants of P. setosum, which are perennial and reproduce vegetatively or by agamospermy, possessed a single specific haplotype. This species differed clearly from the five other morphological species, which are known to be annual, to show either sexual or agamospermic reproduction and which shared most of the 12 other haplotypes observed in the section, suggesting the occurrence of multiple hybridization events between the taxa. Chloroplast DNA variation was highly geographically structured, suggesting low seed dispersal between sites, whereas the substantial haplotype diversity observed in the sites may indicate that agamic reproduction is responsible for the maintenance of distinct genetically isolated clones. Haplotype classification using Wagner's parsimony suggested the occurrence of bidirectional gene flow between the diploids and the polyploids, as reported already in other related apomictic complexes.

Africa, Western↗
PubMed1995

Molecular analysis of 36 mutations at the mouse pink-eyed dilution (p) locus.

Thirty-six radiation- or chemically induced homozygous-lethal mutations at the p locus in mouse chromosome 7 have been analyzed at 17 loci defined by molecular probes to determine the types of lesions, numbers of p-region markers deleted or rearranged, regions of overlap of deletion mutations, and genetic distances between loci. A linear deletion map of the [Myod1, Ldh3]-[Snrpn, Znf127] region has been constructed from the molecular analyses of the p-locus deletions. The utility of these deletions as tools for the isolation and characterization of the genes specifying the neurological, reproductive, and developmental phenotypes genetically mapped to this region will grow as more detailed molecular analyses continue.

Animals↗
PubMed2003

Speciation as a positive feedback loop between postzygotic and prezygotic barriers to gene flow.

Speciation is intimately associated with the evolution of sex-and-reproduction-related traits, including those affecting hybrid incompatibility (postzygotic isolation) and species recognition (prezygotic isolation). Genes controlling such traits are not randomly distributed in the genome but are particularly abundant on the sex chromosomes. However, the evolutionary consequences of the sex linkage of genes involved in speciation have been little explored. Here, we present simulations of a continent-island diploid model that examines the effects of reduced recombination using both autosomal and sex-linked inheritance. We show first that linkage between genes affecting postzygotic and prezygotic isolation leads to a positive feedback loop in which both are strengthened. As species recognition evolves, genes causing hybrid incompatibility will hitchhike along with those improving premating isolation, leading to stronger hybrid incompatibility and thus increased pressure for further preference divergence. Second, we show that this loop effect is generally enhanced by sex linkage, because recombination is eliminated in the heterogametic sex, leading to tighter effective linkage between the two classes of genes and because natural selection is more efficient at sex-linked loci, as recessive alleles are not masked by dominance in the heterogametic sex. Accordingly, hitchhiking can be important in promoting speciation and can also lead to increased postzygotic isolation through adaptive evolution.

Adaptation, Biological↗
PubMed2005

Cytogenetic analysis of three Italian populations of Coregonus lavaretus (Pisces, Salmoniformes) with chromosomal localization of major and minor ribosomal genes, and telomeric repeats.

The European whitefish, Coregonus lavaretus, widely distributed in freshwater of northern Europe and introduced into the major lakes of northern Italy, has been restocked in central Italian lakes. In accordance with current managing practices, a reduced number of spawners contribute to reproduction within each lake and a certain degree of isolation is to be expected between populations from different lakes, resulting in the rapid fixing of chromosomal changes. A detailed survey of three populations from different lakes was carried out using classical and molecular cytogenetic techniques, to verify if specific chromosomal markers are present in the distinct populations. The comparative analysis revealed intraspecific variability of NORs and fixed differences in their number in the three populations. A co-localization of major and minor rRNA genes on one chromosome site was also observed. The original data regarding the chromosome mapping of the (TTAGGG)(n) telomeric repeat obtained in this study, demonstrated their exclusively terminal distribution, and a conspicuous inter-chromosomal variation in the number of repeats. The results are compared with data available for populations from native geographic ranges.

Animals↗