Biomedical subjects

J M Dobrowolski

Publications and source records attributed to J M Dobrowolski.

5 recordsLinked to original sources

PubMed1997

Actin in the parasite Toxoplasma gondii is encoded by a single copy gene, ACT1 and exists primarily in a globular form.

Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by the protozoan parasite Toxoplasma gondii. We have characterized the ACT1 gene and localized the conventional isoform of actin that it encodes within T. gondii. The predicted amino acid sequence of ACT1 was most similar to two other parasite actins, Plasmodium falciparum Pfact-1 (93.1% identical) and Cryptosporidium parvum actin (88.1%): among vertebrate actins, ACT1 was most closely related to the mammalian beta and gamma (83%) actin isoforms. Actin-specific antibodies and fluorescently labeled DNAse I were used to localize actin in T. gondii tachyzoites by immunofluorescence and immunoelectron microscopy. Actin was detected beneath the parasite cell membrane and in clusters scattered within the cytosol of T. gondii tachyzoites. Actin filaments were not detected in detergent-solubilized parasites separated by high speed centrifugation, indicating that actin exists primarily in a globular form in T. gondii.

Actins↗

PubMed1997

Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii.

We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13,400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immunoelectron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.

Actin Depolymerizing Factors↗

PubMed1997

Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum-coated slides. The myosin light-chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii.

Actins↗

PubMed1996

Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite.

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. We demonstrate that invasion is critically dependent on actin filaments in the parasite, but not the host cell. Invasion into cytochalasin D (CD)-resistant host cells was blocked by CD, while parasite mutants invaded wild-type host cells in the presence of drug. CD resistance in Toxoplasma was mediated by a point mutation in the single-copy actin gene ACT1. Transfection of the mutant act1 allele into wild-type Toxoplasma conferred motility and invasion in the presence of CD. We conclude that host cell invasion by Toxoplasma, and likely by related Apicomplexans, is actively powered by an actin-based contractile system in the parasite.

Actin Cytoskeleton↗

PubMed1994

Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus.

Previous studies have established that most if not all of the genes required for synthesis of the Rhodobacter capsulatus essential photosystem are clustered on a 46 kb region of the chromosome known as the photosynthesis gene cluster. This region has recently been sequenced in its entirety by Hearst and co-workers, revealing the existence of 23 open reading frames, many of which are thought to be involved in the synthesis of bacteriochlorophyll. In this study we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis. The results of this analysis demonstrate that mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway. One mutation was observed to result in partial disruption of bacteriochlorophyll biosynthesis, leading to the accumulation of bacteriochlorophyll as well as an intermediate in the biosynthetic pathway. We also observed that disruptions constructed in four open reading frames had no discernible effect on the synthesis of photopigments. The results of this analysis are discussed with regard to our current understanding of the role of each of these open reading frames in the synthesis of the R. capsulatus photosystem.

Bacterial Proteins↗