Molecular cloning of a gene encoding matrix metalloproteinase-like protein from Gnathostoma spinigerum.

The advanced third-stage larvae (aL3) of Gnathostoma spinigerum contain a 24 kDa glycoprotein with diagnostic potential. Immunoscreening with the monoclonal antibody to the 24-kDa protein (mAb GN6/ 24) has identified a cDNA clone with an insert of 932 base pairs (bp). The insert contains a full-length gene of 732 bp encoding a protein that is 33-39% similar to matrix metalloproteinases (MMPs) of Caenorhabditis elegans and several lower and higher vertebrates. The MMP-like protein of G. spinigerum possesses the catalytic domain, but lacks the propeptide and hemopexin-like domains found in other MMPs. A signal peptide of 23 amino acids at its amino terminus indicates that it is a secretory protein, which is confirmed by Western blot analysis showing the presence of the 24 kDa protein in the excretory-secretory products of aL3.

Production of monoclonal antibodies to the cuticle of advanced third-stage larva of Gnathostoma spinigerum.

This study has demonstrated that sera from Balb/c mice infected with live advanced third-stage larvae (aL3), but not those immunized with crude larval extract, immunoprecipitated the 25-kDa protein from surface-iodinated extract of aL3. Hybridoma cell lines derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissue of aL3 including the esophagus, intestine, muscle and cuticle by immunofluorescence assay. However, none of the cuticle-positive hybridoma cell lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. Western blot analysis showed that monoclonal antibodies (MAbs) secreted by clones derived from one of the cuticle-positive hybridoma lines recognized proteins of molecular weights ranging from 55-96 kDa. The MAbs most likely reacted with the collagenous component of the cuticle.

Recognition of deglycosylated larval proteins of Gnathostoma spinigerum by a monoclonal antibody and human gnathostomiasis antiserum.

The study on the recognition of 35S-labelled somatic antigens of Gnathostoma spinigerum advanced third-stage larva (aL3) has revealed that the mAb GN6/24 immunoprecipitated 26- and 24-kDa proteins from the undigested and N-glycosidase F-digested larval extracts, respectively. The recognition of the deglycosylated form of the glycoprotein indicated that the mAb reacted with the peptide epitope on the 26-kDa protein. Human gnathostomiasis antiserum immunoprecipitated most of the N-glycosidase F-digested larval proteins including the deglycosylated 26-kDa protein.

Fluctuations of larval excretion in Strongyloides stercoralis infection.

Follow-up stool examinations were carried out on two groups of the subjects who were screened negative (group 1) or positive (group 2) for Strongyloides stercoralis by the agar plate culture. This technique could detect S. stercoralis larvae in 87.5-96.4% of the subjects in group 2 and 0-5.9% of the subjects in group 1 on various days of the eight-week and four-week follow-up periods, respectively. The detection rate on each day of examination was not statistically different from that on the first day in both groups. Quantitative measurement of S. stercoralis larvae excreted in the feces of the subjects in group 2 by the standard direct smear method of Beaver and others revealed slight to marked fluctuations of the larval output in individual subjects. From the results of both stool examination methods, it could be implied that 52% of S. stercoralis-infected individuals had low-level infection.

Detection of circulating Trichinella spiralis larvae by polymerase chain reaction.

Several investigators have successfully applied the polymerase chain reaction to the amplification of DNA from Trichinella spiralis muscle-stage larvae. We show herein that specific DNA can be amplified from T. spiralis migratory larvae in the blood of experimentally infected mice. The polymerase chain reaction detected the presence of migratory larvae in mouse blood from day 5 to day 14 of infection. The technique may be applied to human trichinosis, but its diagnostic value will depend on the severity and stage of the infection.

Further characterization of a 58 kDa Plasmodium berghei phosphoprotein as a cochaperone.

Molecular chaperones are important for proper protein folding during protein biogenesis. This report describes a protein from Plasmodium berghei which is 30% identical and 40% similar to a recently described mammalian cochaperone, or heat shock protein 70 interacting protein. The P. berghei cochaperone accumulates throughout the trophozoite stage and decreases during the schizont stage. The stage specific expression is consistent with its presumed role in protein folding or protein-protein interactions. The largest difference between the Plasmodium and mammalian sequences is a more extensive domain of imperfect glycine-glycine-methionine-proline (GGMP) tandem repeats in the parasite's cochaperone sequence. Immunofluorescence studies show that the protein is an abundant cytosolic protein of the parasite. However, antibodies raised against the GGMP repeat domain, which is also found in other parasite chaperones, react with both the parasite and host erythrocyte membrane. The reactivity with the host membrane suggests that the parasite exports molecular chaperones into the infected erythrocyte.

Molecular cloning and localization of an abundant novel protein of Plasmodium berghei.

Screening of Plasmodium berghei genomic libraries using DNA insert corresponding to the 3' half of P. falciparum 70-kDa heat shock protein gene identified several abundant clones which represent a novel gene in the parasite. The complete sequence was obtained using an approach based on inverse polymerase chain reaction. Analysis of the deduced amino acid sequence revealed the presence of 19 imperfect repeats of the sequence Gly-Gly-Met-Pro toward the carboxy terminus. Except for the similar sequence repeated seven times in the malarial 70-kDa heat shock protein, the sequence of the cloned gene product is very different. Moreover, the sequence also revealed acidic and basic domains in the protein which are more than 60% similar in sequence to functional domains present in numerous DNA binding transcription factors. A 56-kDa protein was identified by immunoprecipitation from labeled P. berghei extract using antisera raised in mice against gene products expressed in Escherichia coli. The protein is present in all the different life cycle stages of the parasites as revealed by immunoelectron microscopy.

Expression of members of the heat-shock protein 70 family in the exoerythrocytic stages of Plasmodium berghei and Plasmodium falciparum.

Exoerythrocytic stages of Plasmodium berghei cultured in HepG2-A16 hepatoma cells and those of P. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.