Biomedical subjects

T E Nash

Publications and source records attributed to T E Nash.

At least 37 records · Page 2Linked to original sources

PubMed1998

Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

Acetyltransferases↗

PubMed1998

The molecular mechanisms of giardia encystation.

The protozoan parasite Giardia lamblia is transmitted as an environmentally resistant cyst. The encystation process is attracting attention not only from the viewpoint of disease transmission, but also as a model for differentiation. Here, Hugo Luján, Michael Mowatt and Theodore Nash discuss molecular events underlying this process, including the induction of expression and transport of cyst wall proteins and the induction of Golgi-like activity. They also propose that the signal for encystation derives from cholesterol deprivation in the lower small intestine.

Journal Article↗

PubMed1998

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

Animals↗

PubMed1997

Antigenic variation in Giardia lamblia and the host's immune response.

Giardia lamblia, a protozoan parasite of the small intestine of humans and other animals, undergoes surface antigenic variation. The antigens involved belong to a family of variant-specific surface proteins (VSPs), which are unique, cysteine-rich zinc finger proteins. The patterns of infection in humans and animals fail to show the expected cyclical waves of increasing and decreasing numbers of parasites expressing unique VSPs. Nevertheless, changes in VSP expression occur within the population in vivo owing to selection of VSPs by both immune and non-immune mechanisms. After inoculation of a single G. lamblia clone (able to persist in the absence of immune pressure) expressing one VSP (> or = 90%) into mice or humans, the original VSP continues to be expressed until 2 weeks post inoculation (p.i.), when many other VSPs gradually replace it. Selection by immune-mediated processes is suggested because switching occurs at the same time that humoral responses are first detected. In most mouse strains, switching also occurs at about two weeks. Almost all trophozoites are eliminated at three weeks (p.i.), but a barely detectable infection persists over months. In neonatal mice, apparent self-cure is delayed until the sixth or seventh week. Antigenic switching does not occur in adult or neonatal severe combined immunodeficiency disease (SCID) mice, but does occur in neonatal nude mice, thus implicating B-cell-mediated mechanisms in immune switching. Not all VSPs are expressed to the same degree in vivo. Some VSPs appear to be preferentially selected whereas others are eliminated on a non-immune basis. In infections in which immunity does not play a role, such as in SCID mice, and during the first week of infection in immunocompetent mice or gerbils, persisting VSPs are preferentially expressed and maintained whereas non-persisting VSPs are replaced within the first week of infection. The purpose of antigenic variation may be presentation of a wide assortment of VSPs to hosts, increasing the chance of a successful initial infection or reinfection. Immune selection of variants comes into play following biological selection.

Animals↗

PubMed1997

Mechanisms of Giardia lamblia differentiation into cysts.

Microbiologists have long been intrigued by the ability of parasitic organisms to adapt to changes in the environment. Since most parasites occupy several niches during their journey between vectors and hosts, they have developed adaptive responses which allow them to survive under adverse conditions. Therefore, the life cycles of protozoan and helminthic parasites are excellent models with which to study numerous mechanisms involved in cell differentiation, such as the regulation of gene expression, signal transduction pathways, and organelle biogenesis. Unfortunately, many of these studies are very difficult because the conditions needed to elicit developmental changes in parasites remain undetermined in most cases. Recently, several interesting findings were reported on the process of differentiation of Giardia lamblia trophozoites into cysts. G. lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. It belongs to the earliest identified lineage among eukaryotes and therefore offers a unique insight into the progression from primitive to more complex eukaryotic cells. The discovery of a specific stimulus that induces trophozoites to differentiate into cysts, the identification and characterization of encystation-specific molecules, the elucidation of novel biochemical pathways, and the development of useful reagents and techniques have made this parasite an excellent model with which to study differentiation in eukaryotic cells. In this review, we summarize the most recent fundings on several aspects of Giardia differentiation and discuss the significance of these findings within the context of current knowledge in the field.

Animals↗

PubMed1996

Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.

Giardia lamblia, like most human intestinal parasitic protozoa, sustains fundamental morphological and biochemical changes to survive outside the small intestine of its mammalian host by differentiating into an infective cyst. However, the stimulus that triggers this differentiation remains totally undefined. In this work, we demonstrate the induction of cyst formation in vitro when trophozoites are starved for cholesterol. Expression of cyst wall proteins was detected within encystation-specific secretory vesicles 90 min after the cells were placed in lipoprotein-deficient TYI-S-33 medium. Four cloned lines derived from two independent Giardia isolates were tested, and all formed cysts similarly. Addition of cholesterol, low density or very low density lipoproteins to the lipoprotein-deficient culture medium, inhibited the expression of cyst wall proteins, the generation of encystation-specific vesicles, and cyst wall biogenesis. In contrast, high density lipoproteins, phospholipids, bile salts, or fatty acids had little or no effect. These results indicate that cholesterol starvation is necessary and sufficient for the stimulation of Giardia encystation in vitro and, likely, in the intestine of mammalian hosts.

Animals↗

PubMed1996

Increased expression of the molecular chaperone BiP/GRP78 during the differentiation of a primitive eukaryote.

Giardia lamblia, a major cause of intestinal disease worldwide, is a parasitic protozoan that represents the earliest branch of the eukaryotic lineage. Trophozoites, which possess two nuclei but lack mitochondria, peroxisomes and a typical Golgi apparatus, colonize the small intestine of the vertebrate host where they may differentiate into infective cysts. Encystation is a regulated process characterized by the biosynthesis, secretion and formation of a protective extracellular cyst wall. In previous studies, we demonstrated the biogenesis of the Golgi apparatus during encystation and identified two leucine-rich proteins (CWPs), which localize within encystation-specific secretory granules before their incorporation into the cyst wall. Here, we used immunological, biochemical and molecular biological approaches to analyze the expression of BiP/GRP78, an endoplasmic reticulum (ER)-resident chaperone, during the Giardia life cycle. A monoclonal antibody specific for Giardia BiP permitted the visualization of the ER of this protozoan and showed that BiP expression increased simultaneously with the increased expression of CWPs during encystation. However, in contrast to the 140-fold increase in levels of CWP transcripts, the steady-state level of BiP mRNA did not increase during encystation. Furthermore, potent inducers of BiP expression in higher eukaryotic cells, including agents that perturb the ER environment, did not affect BiP expression in Giardia. These results, when considered together with the profound changes that occur in the secretory pathway during Giardia encystation, indicate an important role for this molecular chaperone during the differentiation of this primitive eukaryote.

Animals↗

PubMed1995

Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall.

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

Amino Acid Sequence↗

PubMed1995

Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties.

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

Amino Acid Sequence↗

PubMed1995

Transient transfection and expression of firefly luciferase in Giardia lamblia.

We have developed a gene transfer system for the protozoan parasite Giardia lamblia. This organism is responsible for many cases of diarrhea worldwide and is considered to be one of the most primitive eukaryotes. Expression of a heterologous gene was detected in this parasite after electroporation with appropriate DNA constructs. We constructed a series of transfection plasmids using flanking sequences of the Giardia glutamate dehydrogenase (GDH) gene to drive expression of the firefly luciferase reporter gene. The optimal construct consisted of a GDH/luciferase fusion gene in which the first 18 codons of the GDH gene immediately preceded the luciferase gene; this fusion gene was flanked by the upstream and downstream sequences of the GDH gene. Electroporation of this construct into Giardia yielded luciferase activity that was 3000- to 50,000-fold above background. Removal of either the 5' or 3' GDH flanking sequences from this construct resulted in significantly reduced luciferase activity, and removal of both flanking sequences reduced luciferase activity to background levels. Luciferase activity was proportional to the amount of DNA electroporated and was maximal at 6 hr after electroporation.

Animals↗

PubMed1995

Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia.

A fundamental characteristic of eukaryotic cells is the presence of membrane-bound compartments and membrane transport pathways in which the Golgi complex plays a central role in the selective processing, sorting, and secretion of proteins. The parasitic protozoan Giardia lamblia belongs to the earliest identified lineage among eukaryotes and therefore offers unique insight into the progression from primitive to more complex eukaryotic cells. Here, we report that Giardia trophozoites undergo a developmental induction of Golgi enzyme activities, which correlates with the appearance of a morphologically identifiable Golgi complex, as they differentiate to cysts. Prior to this induction, no morphologically or biochemically identifiable Golgi complex exists within nonencysting cells. Remarkably, protein secretion in both nonencysting and encysting trophozoites is inhibited by brefeldin A, and brefeldin A-sensitive membrane association of ADP-ribosylation factor and beta-COP is observed. These results suggest that the secretory machinery of Giardia resembles that of higher eukaryotes despite the absence of a Golgi complex in nonencysting trophozoites. These findings have implications both for defining the minimal machinery for protein secretion in eukaryotes and for examining the biogenesis of Golgi structure and function.

4-Chloro-7-nitrobenzofurazan↗

PubMed1995

Isoprenylation of proteins in the protozoan Giardia lamblia.

We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.

Acetylcysteine↗

PubMed1995

Developmentally regulated expression of a Giardia lamblia cyst wall protein gene.

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100-fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation-specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine-rich polypeptide of M(r) 26,000 that contains 5.3 tandemly arranged copies of a degenerate 24-amino-acid repeat. A hydrophobic amino-terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

Amino Acid Sequence↗

PubMed1994

Insulin-like growth factors stimulate growth and L-cysteine uptake by the intestinal parasite Giardia lamblia.

Giardia lamblia, a parasitic protozoan responsible for diarrhea and malabsorption in humans, grows axenically only in media that contain serum and a high concentration of L-cysteine. During our attempts to grow Giardia in the absence of serum, we found that: (a) human insulin-like growth factors (especially IGF-II), but not insulin, promote the growth and L-cysteine uptake by G. lamblia trophozoites; (b) the growth stimulation was inhibited by alpha IR3, an anti-type 1 IGF receptor monoclonal antibody, but an anti-type 2 IGF receptor antibody had no effect; and (c) IGFs act on Giardia through a type 1 IGF receptor-like protein, which can bind IGF-II with higher affinity than IGF-I, and most likely possesses intrinsic phosphotyrosine kinase activity.

Animals↗

PubMed1994

Predictors of upper gastrointestinal bleeding in patients with schistosomal periportal fibrosis.

A case-control study was conducted between 1985 and 1987 in the Gezira-Managil area of central Sudan to assess the major predictors of haematemesis. Eighty-four patients who had suffered at least one attack of oesophageal bleeding and had schistosomal periportal fibrosis demonstrated by ultrasonography were compared with 173 subjects without bleeding but with ultrasonographic evidence of periportal fibrosis. A splenic longitudinal dimension of more than 11 cm, periportal fibrosis worse than grade I and varices more than grade I were independently associated with a significant risk of variceal bleeding. Age, sex, presence of a palpable liver and portal vein diameter were not associated with a significant risk of bleeding after adjustment for potential confounding variables. Factors identified in this study could be helpful in the prophylactic management of patients with complicated schistosomiasis.

Adult↗

PubMed1994

Complementation of an Escherichia coli glycolysis mutant by Giardia lamblia triosephosphate isomerase.

The Giardia lamblia triosephosphate isomerase (TIM) gene was cloned using a probe generated by a polymerase chain reaction that employed primers complementary to highly conserved regions of TIM. The nucleotide sequence predicts a protein that is 38 and 47% identical to TIM from prokaryotic and eukaryotic sources, respectively. Like all other Giardia protein-coding genes studied thus far, the TIM gene lacks introns and is transcribed to yield a polyadenylated mRNA with an extremely short 5' untranslated region. The Giardia TIM gene complemented an Escherichia coli triosephosphate isomerase deletion mutant. The simplicity and success of complementation suggests its general utility in cloning Giardia genes of known function.

Amino Acid Sequence↗

PubMed1994

Serum Cohn fraction IV-1 supports the growth of Giardia lamblia in vitro.

Bovine serum Cohn fractions were substituted for whole bovine serum in TYI-S-33 medium. Only fraction IV-1 supported attachment, cysteine uptake, and growth of Giardia lamblia. Among the mammalian sources tested, only goat and horse fractions showed activities comparable to that of the bovine fraction. Agglutinating immunoglobulins were identified as deleterious agents in human, rat, rabbit, and dog fractions.

Animals↗

PubMed1994

Size heterogeneity among antigenically related Giardia lamblia variant-specific surface proteins is due to differences in tandem repeat copy number.

Giardia lamblia undergoes antigenic variation by modulating the expression of the different genes that comprise the trophozoite's variant-specific surface protein (VSP) repertoire. We studied an epitope that is conserved among VSPs expressed by cloned trophozoite lines derived from the independent G. lamblia isolates WB, G3M, Be-2, and CAT. The epitope recognized by monoclonal antibody 6E7 lies entirely within the region of tandemly repeated 65-amino-acid units that is characteristic of these size-variant VSPs. Northern (RNA) hybridization, cDNA cloning, and DNA sequence analysis indicate that size heterogeneity among these VSPs is due to differences in the number of repetitive units.

Amino Acid Sequence↗