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Biomedical subjects

D J Kemp

Publications and source records attributed to D J Kemp.

At least 55 records · Page 3Linked to original sources
PubMed1994

Clonal diversity of Streptococcus pyogenes within some M-types revealed by multilocus enzyme electrophoresis.

Twenty-two reference isolates and 30 local isolates of group A Streptococci were classified into 36 electrophoretic types (ET) on the basis of allozyme variation at 27 enzyme loci. Local isolates were characterized by a high frequency of M-non typable strains. M-type and ET were more closely associated in local isolates from an endemically-infected population; nevertheless, amongst the local isolates there were also strains of the same ET type with different M-types. A possible explanation is that genetic exchange between strains may introduce different M-types into strains of defined ET when these are exposed to strong selection in the presence of heavy loads of infection. In contrast to the reported clustering of strains associated with toxic shock-like syndrome into two closely related ET clones, we found no relationship of ET phenotype to acute poststreptococcal glomerulonephritis or rheumatic fever.

Alleles↗
PubMed1994

Protective immunity induced in squirrel monkeys with recombinant apical membrane antigen-1 of Plasmodium fragile.

Saimiri sciureus boliviensis monkeys were immunized with the Plasmodium fragile form of the merozoite apical membrane antigen-1 produced using the baculovirus expression system and combined with Montanide ISA 720 adjuvant. Following three immunizations, monkeys were challenged with 10,000 P. fragile trophozoite parasites. Antibody titers determined by fluorescence microscopy indicated an enhanced response following the second immunization. Four of five control animals had parasite counts > 5% 18-26 days following challenge. Four of five immunized monkeys had reduced levels of maximum parasitemia or delays in accumulated parasite counts, suggestive of protection. Rechallenge of the animals with P. falciparum resulted in three of four adjuvant control animals developing patent parasitemia whereas none of five immunized animals were infected, suggesting some level of heterologous protection.

Adjuvants, Immunologic↗
PubMed1993

Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.

Agglutination↗
PubMed1993

A Plasmodium chabaudi antigen located in the parasitophorous vacuole membrane.

In order to study antigens from the rodent malaria Plasmodium chabaudi, clones reacting with mouse hyperimmune serum were selected from an expression library of blood-stage P. chabaudi cDNAs in the vector pGEX-2T. Sixty-four such clones were shown to derive from 19 different P. chabaudi antigens. One of these, Antigen 3008 (Ag3008), has a predicted size of 17.5 kDa and an observed size of 24 kDa. It is located in the parasitophorous vacuole membrane of maturing parasites and in the dense granules of merozoites. The cDNA sequence predicts a highly charged molecule with an N-terminal signal sequence and a central transmembrane domain, but no tandem repeats. The sequence reported for Pc24, a previously identified but uncharacterized P. chabaudi protein, corresponds to part of the 3' untranslated region in the Ag3008 mRNA. Ag3008 has many features in common with the P. falciparum circumsporozoite protein-related antigen (CRA).

Amino Acid Sequence↗
PubMed1992

Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Cloning and analysis of the RESA-2 gene: a DNA homologue of the ring-infected erythrocyte surface antigen gene of Plasmodium falciparum.

We have cloned and sequenced a homologue of the ring-infected erythrocyte surface antigen (RESA) gene from Plasmodium falciparum designated RESA-2. Two reading frames with high homology to exon 1 and exon 2 of RESA at both the nucleotide and amino acid levels were identified in the RESA-2 sequence. However, RESA-2 does not contain either of the blocks of tandem repeats present in RESA. The lack of an RNA transcript in either asexual or sexual stage parasites and the presence of an in-frame stop codon in the second reading frame suggests RESA-2 could be a pseudogene. Its lack of expression in asexual stages demonstrates that it does not complement the RESA deletion in isolate FCR3.

Amino Acid Sequence↗
PubMed1992

Towards a high-resolution map of the Plasmodium falciparum genome.

Until recently very little was known about the genome of Plasmodium falciparum. The situation has changed considerably with the advent of pulsed field gradient electrophoresis and yeast artificial chromosome technologies. It should now be possible to generate a high-resolution map within a few years. Here, Tony Triglia, Thomas Wellems and David Kemp review current knowledge.

Journal Article↗
PubMed1992

A chromosome 9 deletion in Plasmodium falciparum results in loss of cytoadherence.

Many lines of Plasmodium falciparum undergo a deletion of the right end of chromosome 9 during in vitro culture accompanied by loss of cytoadherence and gametocytogenesis. Selection of cytoadherent cells from a mixed population co-selects for those with an undeleted chromosome 9 and the selected cells produce gametocytes. The deletion also results in loss of expression of PfEMP1, the putative cytoadherence ligand, suggesting that PfEMP1 or a regulatory gene controlling PfEMP1 expression and gametocytogenesis may be encoded in this region. We have isolated several markers for the deleted region and are currently using a YAC-P. falciparum library to investigate this region of the genome in detail.

Animals↗
PubMed1991

Structural diversity in the Plasmodium falciparum merozoite surface antigen 2.

Antigens associated with the surface of merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known merozoite surface antigens (merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved N- and C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those defined in known MSA-2 sequences.

Amino Acid Sequence↗
PubMed1991

Large fragments of Plasmodium falciparum DNA can be stable when cloned in yeast artificial chromosomes.

A major problem in cloning large segments of Plasmodium falciparum DNA is the instability of this very A + T-rich DNA in Escherichia coli. We have therefore investigated whether P. falciparum DNA segments over 100 kb in size cloned in the yeast Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) are stable. An analysis of EcoRI fragments adding up to 230 kb, or nearly 1% of the P. falciparum genome, showed that sequences cloned as YACs were indistinguishable from the DNA in the P. falciparum genome, showing neither deletions nor rearrangements. Over 400 YACs have been obtained so far with an average insert size between 110 and 130 kb. Screening the YACs with single-copy probes suggested that most sequences should be represented in a library of less than 10(3) YACs. Hence YACs provide an approach to cloning large segments of P. falciparum DNA.

Animals↗
PubMed1991

7H8/6, a multicopy DNA probe for distinguishing isolates of Plasmodium falciparum.

A method using DNA fingerprinting to distinguish Plasmodium falciparum isolates has been developed using a clone, 7H8/6, isolated by screening a genomic expression library of the Malayan Camp strain with a monoclonal antibody. Unlike other P. falciparum DNA probes that contain internally repetitive sequences, this probe contains a single full-length open reading frame and lacks any repetitive sequences. Chromosome mapping located the hybridizing sequences to most chromosomes. 7H8/6 sequences are located at sites near the subtelomeric regions, although they are further from the telomeres than the subtelomeric repetitive sequences rep20. The probe gave multiple polymorphic bands on Southern blots of genomic DNA allowing all unrelated isolates tested to be readily distinguished. Hybridization patterns of parent and progeny from the genetic cross of two P. falciparum clones showed that most bands were inherited independently and no new bands were generated during the cross. This probe is useful for distinguishing isolates of P. falciparum and also for analyzing relationships between closely related isolates.

Animals↗
PubMed1991

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.

Animals↗
PubMed1991

Immunization of owl monkeys with the ring-infected erythrocyte surface antigen of Plasmodium falciparum.

Aotus nancymai were immunized with the 4-mer, 8-mer, and 11-mer repeat peptides of the ring-infected erythrocyte surface antigen molecule of Plasmodium falciparum conjugated to diphtheria toxoid with muramyl dipeptide (MDP) as adjuvant. Immunization failed to induce protective immunity against the Uganda Palo Alto strain of P. falciparum as judged by maximum levels of parasitemia of immunized monkeys relative to those of controls. The fused polypeptide FPAg632, when combined with MDP, also failed to induce protective immunity. However, the maximum level of parasitemia and serologic response to the 11-mer peptide were inversely correlated. The safety of the use of MDP was evident.

Acetylmuramyl-Alanyl-Isoglutamine↗