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Biomedical subjects

M L Crouch

Publications and source records attributed to M L Crouch.

14 recordsLinked to original sources
PubMed2001

Binding of fibronectin by Trichomonas vaginalis is influenced by iron and calcium.

We have reported that Trichomonas vaginalis, the causative agent of the most common, non-viral sexually transmitted disease, bound to cover slips coated with fibronectin (FN) (Crouch & Alderete, Microbiol 1999 145: 2835-43). In this study, we extend that observation by showing that FN binding is specific, and we present data on the requirements of FN binding by T. vaginalis. Immunofluorescence and immuno-gold labelling readily detected FN throughout the trichomonal surface. Parasites bound to(125)I-labelled FN in a time- and concentration-dependent fashion. In the absence of protease inhibitor, iodinated FN was released from the trichomonad surface. Unlabelled FN specifically competed for binding in a concentration-dependent fashion with the(125)I-labelled FN. Interestingly, the amount of FN bound by T. vaginalis organisms was dependent on iron. High-iron-grown trichomonads acquired lower numbers of molecules but with 10-fold higher affinity than low-iron-grown organisms. Further, we show that for iron-replete organisms, calcium (Ca(2+)) at physiological levels increased amounts of bound FN. The increase in binding was rapid, occurring within 5 min of Ca(2+)addition, and required de novo protein synthesis. Co-incubation of live parasites with Ca(2+)in the presence of FN was necessary to increase the amount of FN bound. Treatment of trichomonads with okadaic acid, but not other phosphatase inhibitors, resulted in a 50% decrease in binding of FN, regardless of the presence of Ca(2+), suggesting a role for phosphatase in FN association. These results indicate that depending on the iron status of T. vaginalis organisms in vivo, Ca(2+)may influence trichomonad recognition and binding to FN during host parasitism.

Animals↗
PubMed1999

Trichomonas vaginalis interactions with fibronectin and laminin.

The sexually transmitted protozoan Trichomonas vaginalis cytoadheres to vaginal epithelial cells and causes contact-dependent cytotoxicity which, when combined with the normal exfoliation process, leads to erosion of the epithelium, which may allow trichomonads into extracellular matrix and basement membrane sites. Therefore, the association of T. vaginalis with immobilized fibronectin (FN) and laminin (LM) on cover-slips was examined. Binding of live parasites to coated cover-slips was time- and parasite-density-dependent. Coincubation with an inhibitor of trichomonad cysteine proteinases resulted in an increased attachment of parasites to FN but had no effect on binding to LM, denoting that protease activity influenced optimal FN associations. Further, 20 h mid-exponential phase trichomonads placed in fresh culture medium for 3 h gave higher levels of binding to FN, suggesting that changes during growth in vitro to T. vaginalis organisms affect maximal levels of binding to FN. Extended incubation with substrates diminished the capacity of parasites to bind FN and LM. Treatment of live organisms with periodate reduced binding to LM but not FN, suggesting a role for carbohydrates. In addition, trypsinization of live parasites decreased numbers bound to both substrates. Placement of trypsinized parasites in medium for 2 h fully regenerated binding to FN but not LM. Incubation of trypsinized parasites with cycloheximide abrogated regeneration of attachment to FN, affirming a role for synthesized surface proteins in FN binding. Importantly, the T. vaginalis adhesin proteins that mediate cytoadherence, and iron, a factor that regulates adhesin synthesis, were not involved in FN and LM recognition. These results suggest a role for surface proteins and carbohydrates in trichomonal associations with FN and LM, respectively.

Animals↗
PubMed1992

Molecular analysis of a cruciferin storage protein gene family of Brassica napus.

We have isolated a five-member gene subfamily which encodes cruciferin, a legumin-like 12S storage protein of Brassica napus L., and have analyzed the structure and expression of the family members in developing embryos. Sequence analysis has shown that the coding regions of all five genes are highly similar, with the two most divergent members of the family retaining 89% sequence identity. The analysis of this cruciferin gene family's expression indicates that the developmental pattern of expression of each gene is similar, and the steady-state mRNA levels of each gene are approximately equivalent to each other at all developmental stages.

Allergens↗
PubMed1991

Differential expression of members of the napin storage protein gene family during embryogenesis in Brassica napus.

S1 nuclease analysis and sub-family-specific oligonucleotide probes were used to characterize the expression during embryogenesis of the napin storage protein gene family of Brassica napus (oilseed rape). The expression of one sub-class represented by the napin gene gNa peaks and declines earlier than the other members of the family. This sub-class was highly expressed representing ca. 20% of napin mRNA at 26 days after anthesis.

2S Albumins, Plant↗
PubMed1991

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos.

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.

2S Albumins, Plant↗
PubMed1990

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.

Amino Acid Sequence↗
PubMed1989

Seed Storage Protein Transcription and mRNA Levels in Brassica napus during Development and in Response to Exogenous Abscisic Acid.

Transcription rates and mRNA levels for Brassica napus seed storage protein families, cruciferin and napin, have been determined in embryos developing in the seed, as well as in embryos cultured with and without abscisic acid. Cruciferin and napin mRNAs are high during the cell expansion phase of embryo development, representing as much as 11 and 8%, respectively, of the total embryo mRNA. During the same time cruciferin and napin gene transcription rates, as measured in isolated nuclei, are also high. The data indicate that cruciferin mRNA is more stable than napin mRNA because while the napin transcription rate is higher than the cruciferin transcription rate, the cruciferin mRNA accumulates to higher levels. However, late in embryo development, both cruciferin and napin mRNAs seem to be less stable than earlier because comparable transcription rates result in lower mRNA levels. When embryos are cultured in the presence of abscisic acid, the levels of cruciferin and napin mRNAs are two- to threefold higher than in embryos cultured on basal medium. The transcription rates show a similar increase in the presence of abscisic acid, suggesting that abscisic acid is responsible for the increased mRNA level at least in part through an increase in the transcription rate of the two genes.

Journal Article↗
PubMed1988

Regulating recombinant DNA biologics.

Biologics have been regulated in the United States since 1902. Many new products and technologies have been introduced since that original Act defined a biologic and specified how they should be controlled. Fortunately, that authorizing authority, now known as Section 351 of the Public Health Service Act, proved to be flexible enough to provide adequate assurance of the safety, purity, and efficacy of these new products, with minimal promulgation of regulations. The new biotechnology products, although presenting a challenge, are being regulated consistent with our policy for all other biologics. The scientific advances now possible because of recombinant DNA technology are seeming unlimited. Safety issues resulting from the use of such products are examined. These issues include: a) alterations in molecular structure; b) chemical and physical alterations; and c) purity standards of final products. Lastly, strategies that have been developed for regulating these biotechnical products, within the framework of FDA statutes and regulations are reviewed.

Biotechnology↗
PubMed1987

Nucleotide sequence of a member of the napin storage protein family from Brassica napus.

We have begun the molecular characterization of genes encoding napin, the 1.7 S embryo-specific storage protein of Brassica napus. Genomic Southern blot analysis indicates that napin is encoded by a multigene family comprised of a minimum of 16 genes. Two DNA fragments containing single napin genes have been recovered from B. napus genomic libraries. We have determined the nucleotide sequence of one member of the napin gene family, gNa. The gene has a simple structure lacking introns and containing the canonical features expected for genes transcribed by RNA polymerase II. The site of the initiation of transcription was determined to be 37 base pairs upstream of the initiation codon by S1 and primer extension analyses. A gene-specific hybridization probe from the 3' non-translated portion of gNa was used to demonstrate transcription of gNa.

2S Albumins, Plant↗
PubMed1986

Rapeseed embryo development in culture on high osmoticum is similar to that in seeds.

The development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without high osmoticum (0.48 and 0.69 molar sorbitol) was compared with normal development in situ to investigate the role of a drying environment in embryo maturation. Sensitivity to osmoticum was assayed in terms of its ability to mimic normal development, i.e. to both suppress germination and maintain 12 S storage protein (cruciferin) synthesis at levels comparable to those seen in the developing seed. The osmotic conditions used block germination of predesiccation stage embryos but were not sufficient to prevent desiccation stage embryos from taking up water and germinating. At all stages tested, the osmotically treated embryos had approximately normal levels of cruciferin mRNA. Measurements of endogenous abscisic acid (ABA) levels by radioimmunoassay indicated that the osmotic effects on germination and gene expression were not mediated by elevated embryonic ABA. Comparison of the kinetics of osmotic and ABA effects on gene expression showed that the osmotic effect is more rapid. These results are consistent with the hypothesis that ABA acts by inhibiting water uptake, which mechanically prevents germination and affects gene expression in some unknown manner.

Journal Article↗
PubMed1985

Role of ABA in Maturation of Rapeseed Embryos.

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.

Journal Article↗
PubMed1983

cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor polypeptide.

Napins are a family of small, basic storage proteins synthesized in Brassica napus (rapeseed) embryos during seed maturation. Cultured embryos also synthesize napins but require exogenous abscisic acid (ABA) to maintain high accumulation rates. We synthesized cDNA from total RNA of embryos cultured on a medium containing ABA, and cloned it into the Pst1 site of pBR322. Two clones containing napin cDNA sequences selected by differential colony hybridization using [32P]cDNA probes from embryos grown with or without ABA were analyzed. These clones, pN1 (insert size = 583 bp) and pN2 (insert size = 739 bp), contained cDNA from two different napin mRNAs. The mRNAs to which they hybridized were found to encode a 21,000-dalton polypeptide that was immunoprecipitated by antibodies to mature napin (subunits of 9,000 and 4,000 daltons). The cDNA clones hybridized to an 850-base mRNA. Nucleotide sequencing demonstrated 95% homology between pN1 and pN2 cDNA inserts and predicted a precursor polypeptide of 178 amino acids, consistent with the 21,000 dalton in vitro translation product. Comparison of the deduced amino acid sequence with published amino acid compositions of mature napin subunits suggests that both the large and the small subunits are present in one precursor polypeptide, and that other regions of the precursor are removed during processing.

Amino Acid Sequence↗