Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.

Integrins: a family of cell surface receptors.

Activated protein C (APC) is a serine protease with potent anticoagulant properties, which is formed in blood on the endothelium from an inactive precursor During normal haemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa (ref 2) To do this efficiently the enzyme needs a nonenzymatic cofactor, protein S (ref 3) Recently it was found that the anticoagulant response to APC (APC resistance) was very weak in the plasma of 21% of unselected consecutive patients with thrombosis and about 50% of selected patients with a personal or family history of thrombosis; moreover, 5% of healthy individuals show APC resistance, which is associated with a sevenfold increase in the risk for deep vein thrombosis Here we demonstrate that the phenotype of APC resistance is associated with heterozygosity or homozygosity for a single point mutation in the factor V gene (at nucleotide position 1,691, G-->A substitution) which predicts the synthesis of a factor V molecule (FV Q506, or FV Leiden) that is not properly inactivated by APC The allelic frequency of the mutation in the Dutch population is approximately 2% and is at least tenfold higher than that of all other known genetic risk factors for thrombosis (protein C (ref 8), protein S (ref 9), antithrombin10 deficiency) together

/pdf/mutation-in-blood-coagulation-factor-v-associated-with-33avp1ullq.pdf

Mutation in blood coagulation factor V associated with resistance to activated protein C

Publisher Summary Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.

Biology of natural killer cells.

The integrated processing of signals transduced by activating and inhibitory cell surface receptors regulates NK cell effector functions. Here, I review the structure, function, and ligand specificity of the receptors responsible for NK cell recognition.

Nk cell recognition

A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences

Plasmodium falciparum causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 μM for the DHPS allele from sensitive isolates to 112 μM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides.

https://www.pnas.org/content/pnas/94/25/13944.full.pdf

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum

Plasmodium falciparum causes the most se- vere form of malaria in humans. An important class of drugs in malaria treatment is the sulfoneysulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resis- tance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of P. falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity. Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 mM for the DHPS allele from sensitive isolates to 112 mM for an enzyme expressed in a highly resistant isolate. Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P. falciparum are central to the mechanism of resistance to sulfones and sulfonamides. The sulfonamideysulfone group of compounds is used exten- sively in the treatment of bacterial diseases including infections caused by Streptococcus spp., Neisseria spp., and Mycobacte- rium spp as well as many parasitic infections involving Pneu- mocystis, Toxoplasma, Cryptosporidium, and Plasmodium. Members of this group of chemotherapeutic compounds, such as sulfadoxine and dapsone, inhibit the enzyme dihydrop- teroate synthase (DHPS) (1), a component of the folate biosynthetic pathway. The enzyme DHPS catalyzes the con- densation of p-aminobenzoic acid (pABA) with 6-hydroxy- methyldihydropterin pyrophosphate to yield 7,8-dihydrop- teroate (1). Plasmodia generate the vast majority of their folates de novo, and hence inhibition of DHPS by these drugs leads to depletion of dTTP and decreased DNA synthesis (2). However, there is also some evidence for folate salvage by the parasite (3). The human host lacks most of this biosynthetic pathway, and consequently many effective chemotherapeutic agents have been developed that target some of the enzymes involved in the synthesis of pyrimidines. In the case of Plasmodium falciparum, the causative agent of the most lethal form of human malaria, sulfadoxine has been used extensively for prophylaxis. Usually, this has been in combination with the antifolate drug, pyrimethamine, which inhibits the enzyme dihydrofolate reductase (DHFR) (4), because the combination of these drugs shows a marked synergism in their antimalarial effect (5). These drugs have

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum (malariaysulfadoxineydrug resistance)

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2000.02175.x

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.

Tony Triglia

Papers

A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum

Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum (malariaysulfadoxineydrug resistance)

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum