scholarly journals The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant.

1992 ◽  
Vol 12 (3) ◽  
pp. 1194-1201 ◽  
Author(s):  
R D Adam ◽  
Y M Yang ◽  
T E Nash
Keyword(s):  
Giardia Lamblia ◽  
Antigenic Variant ◽  
Amino Acid Homology ◽  
Variable Expression ◽  
Cysteine Rich Protein ◽  
Pair Sequence ◽  
Terminal Amino ◽  
Membrane Anchoring

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.

scholarly journals The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant

1992 ◽  
Vol 12 (3) ◽  
pp. 1194-1201
Author(s):  
R D Adam ◽  
Y M Yang ◽  
T E Nash
Keyword(s):  
Giardia Lamblia ◽  
Antigenic Variant ◽  
Amino Acid Homology ◽  
Variable Expression ◽  
Cysteine Rich Protein ◽  
Pair Sequence ◽  
Terminal Amino ◽  
Membrane Anchoring

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

scholarly journals A synthetic glucagon-like peptide-1 analog with improved plasma stability

1998 ◽  
Vol 159 (1) ◽  
pp. 93-102 ◽  
Author(s):  
U Ritzel ◽  
U Leonhardt ◽  
M Ottleben ◽  
A Ruhmann ◽  
K Eckart ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasma Insulin ◽  
Rat Plasma ◽  
Plasma Stability ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1) is the most potent endogenous insulin-stimulating hormone. In the present study the plasma stability and biological activity of a GLP-1 analog, [Ser]GLP-1(7-36)amide, in which the second N-terminal amino acid alanine was replaced by serine, was evaluated in vitro and in vivo. Incubation of GLP-1 with human or rat plasma resulted in degradation of native GLP-1(7-36)amide to GLP-1(9-36)amide, while [Ser]GLP-1(7-36)amide was not significantly degraded by plasma enzymes. Using glucose-responsive HIT-T15 cells, [Ser]GLP-1(7-36)amide showed strong insulinotropic activity, which was inhibited by the specific GLP-1 receptor antagonist exendin-4(9-39)amide. Simultaneous i.v. injection of [Ser]GLP-1(7-36)amide and glucose in rats induced a twofold higher increase in plasma insulin levels than unmodified GLP-1(7-36)amide with glucose and a fivefold higher increase than glucose alone. [Ser]GLP-1(7-36)amide induced a 1.5-fold higher increase in plasma insulin than GLP-1(7-36)amide when given 1 h before i.v. application of glucose. The insulinotropic effect of [Ser]GLP-1(7-36)amide was suppressed by i.v. application of exendin-4(9-39)amide. The present data demonstrate that replacement of the second N-terminal amino acid alanine by serine improves the plasma stability of GLP-1(7-36)amide. The insulinotropic action in vitro and in vivo was not impaired significantly by this modification.

Transcription of spacer sequences flanking the rat 45S ribosomal DNA gene

1987 ◽  
Vol 7 (1) ◽  
pp. 314-325
Author(s):  
C A Harrington ◽  
D M Chikaraishi
Keyword(s):  
Ribosomal Dna ◽  
Transcriptional Activity ◽  
Tandem Repeats ◽  
Initiation Site ◽  
Rrna Synthesis ◽  
Rna Transcripts ◽  
Pair Sequence ◽  
Polymerase I

The transcriptional activity of spacer sequences flanking the rat 45S ribosomal DNA (rDNA) gene were studied. Nascent RNA labeled in in vitro nuclear run-on reactions hybridized with both 5' and 3' spacer regions. The highest level of hybridization was seen with an rDNA fragment containing tandem repeats of a 130-base-pair sequence upstream of the 45S rRNA initiation site. Synthesis of RNA transcripts homologous to this internally repetitious spacer region was insensitive to high levels of alpha-amanitin, suggesting that it is mediated by RNA polymerase I. Analysis of steady-state RNA showed that these transcripts were present at extremely low levels in vivo relative to precursor rRNA transcripts. In contrast, precursor and spacer run-on RNAs were synthesized at similar levels. This suggests that spacer transcripts are highly unstable in vivo; therefore, it may be the process of transcription rather than the presence of spacer transcripts that is functionally important. Transcription in this upstream rDNA region may be involved in regulation of 45S rRNA synthesis in rodents, as has been suggested previously for frog rRNA. In addition, the presence of transcriptional activity in other regions of the spacer suggests that some polymerase I molecules may transcribe through the spacer from one 45S gene to the next on rodent rDNA.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 328-333 ◽  
Author(s):  
Elizabeta Nemeth ◽  
Gloria C. Preza ◽  
Chun-Ling Jung ◽  
Jerry Kaplan ◽  
Alan J. Waring ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Progressive Decrease ◽  
Cellular Iron ◽  
Gfp Fusion Protein ◽  
N Terminus ◽  
A Cell ◽  
Terminal Amino ◽  
Peptide Treatment

Abstract Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

scholarly journals Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

2020 ◽  
Vol 75 (5) ◽  
pp. 1218-1227
Author(s):  
Samantha A Michaels ◽  
Han-Wei Shih ◽  
Bailin Zhang ◽  
Edelmar D Navaluna ◽  
Zhongsheng Zhang ◽  
...  
Keyword(s):  
Giardia Lamblia ◽  
Trna Synthetase ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Cell Assay ◽  
Mouse Infection Model ◽  
Mouse Infection ◽  
Methionyl Trna Synthetase

Abstract Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

1990 ◽  
Vol 10 (6) ◽  
pp. 2801-2808 ◽  
Author(s):  
D T Mooney ◽  
D B Pilgrim ◽  
E T Young
Keyword(s):  
Point Mutations ◽  
Wild Type ◽  
Urea Treatment ◽  
Amino Terminal ◽  
In Vitro Processing ◽  
The Matrix ◽  
Terminal Amino ◽  
Processing Protease

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

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