Purification and characterization of DNA polymerases from Plasmodium falciparum.

Fractionation of Plasmodium falciparum cellular extracts by fast protein liquid chromatography (FPLC) identified at least two different DNA polymerases. An aphidicolin-sensitive activity co-purified with a primase activity. This, in combination with other characteristics (processivity, sensitivity to other inhibitors), most likely classifies this enzyme as an alpha-like DNA polymerase. It was, however, relatively resistant to N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (IC50 = 6.6 microM) and differs in this aspect from the host homologue, possibly indicating structural differences between host and parasite DNA polymerase alpha. The other DNA polymerase matched eukaryotic DNA polymerase gamma in all properties tested.

Inhibition of the growth of Plasmodium falciparum and Plasmodium berghei by the DNA polymerase inhibitor HPMPA.

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [HPMPA] belongs to a class of nucleoside analogues originally described as having potent activity against a broad spectrum of DNA viruses. We examined the effects of this class of drugs on the growth of cultured Plasmodium falciparum. Strong inhibition was observed by HPMPA (ID50 = 47 nM) at concentrations more than 1000-fold less than the cytotoxic dose for human cells. 3-deaza-HPMPA was even more strongly inhibitory (ID50 = 8 nM), whereas several other acyclic nucleosides were not effective. In mice infected with Plasmodium berghei, increase of parasitaemia can be blocked for 4-6 days by a single injection of HPMPA. Repeated drug administration blocks parasite growth for prolonged periods at doses that are clinically feasible. We also determined the inhibition of several purified Plasmodium DNA polymerases by diphosphorylated HPMPA (HPMPApp). DNA polymerase alpha-like enzymes of P. falciparum and P. berghei are inhibited with an IC50 = 40 microM and a gamma-like DNA polymerase from P. falciparum is even 40-fold more sensitive to the drug. The inhibition by HPMPApp is competitive with dATP, strongly suggesting that Plasmodium DNA polymerases are targets for this class of nucleotide analogue.

Purification and characterization of DNA polymerases from Plasmodium berghei.

DNA polymerases from the malaria parasite Plasmodium berghei were purified more than 50-fold. Several distinct enzymatic activities were isolated that could be distinguished by the use of various specific DNA polymerase inhibitors. In particular, subdivision into an aphidicolin-sensitive and an aphidicolin-resistant group was possible. Further analysis allowed a better comparison with host DNA polymerases and indicated that one aphidicolin-sensitive DNA polymerase resembled DNA polymerase alpha displaying processive DNA synthesis and using RNA primers, whereas another aphidicolin-sensitive DNA polymerase was distributive and only used DNA primers. Marked differences from the host enzymes do exist, however, such as insensitivity to BuPdGTP. Another P. berghei DNA polymerase was isolated that showed characteristics of a DNA polymerase beta-like enzyme, but which differed from host DNA polymerase beta in its insensitivity to dideoxynucleotides.

Bronchodilator and antiulcer phenoxypyrimidinones.

Series of 5-phenoxy-2(1H)-pyrimidinones, 5-phenoxy-4(3H)-pyrimidinones, and related compounds were prepared in a follow-up of a lead prepared as a potential cyclic nucleotide regulating agent. Compounds were evaluated for bronchodilator activity in histamine-challenged guinea pigs and for anticulcer activity in a cold-restraint, stressed rat ulcer model. Bronchodilator activity comparable to, or greater than, that of theophylline was found in both the 2(1H)- and 4(3H)-pyrimidinone series and was most prominent in analogues containing either an electron-withdrawing or -donating substituent in the para position of the phenoxy ring. Significant antiulcer activity was observed only in the 2(1H)-pyrimidinone series among three closely related analogues. One of these, 5-(m-methylphenoxy)-2(1H)-pyrimidinone (3), exhibited more potent antiulcer effects than the clinically useful antiulcer agent carbenoxolone, without demonstrating bronchodilator activity.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S.

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

Heteroallelism at the het-c locus contributes to sexual dysfunction in outcrossed strains of Neurospora tetrasperma.

Neurospora tetrasperma is naturally heterokaryotic, with cells possessing haploid nuclei of both a and A mating types. As a result, isolates are self-fertile (pseudohomothallic). Occasional homokaryotic ascospores and conidia arise, however, and they produce strains that are self-sterile and must outcross to complete sexual reproduction. Invariably, laboratory crosses employing sibling a and A strains from the same parental heterokaryon restore the pseudohomothallic, heterokaryotic state. In contrast, outcrosses employing a and A strains from different wild isolates typically result in sexual dysfunction. Diverse sexual dysfunction types have been observed, ranging from complete sterility to reduced viability. We report that one type of dysfunction, characterized by spontaneous loss of the heterokaryotic state upon ascospore germination, can result from the interaction of incompatible alleles at heterokaryon incompatibility loci. Specifically, we demonstrate that homoallelism at the het-c locus in N. tetrasperma is required for heterokaryon stability. Heterokaryon incompatibility therefore provides an obstacle to outcrossing in this species, an observation with important implications for fungal life-cycle evolution.

Analysis of the role of interfacial tryptophan residues in controlling the topology of membrane proteins.

Tryptophans have a high affinity for the membrane-water interface and have been suggested to play a role in determining the topology of membrane proteins. We investigated this potential role experimentally, using mutants of the single-spanning Pf3 coat protein, whose transmembrane topologies are sensitive to small changes in amino acid sequence. Mutants were constructed with varying numbers of tryptophans flanking the transmembrane region and translocation was assessed by an in vitro translation/translocation system. Translocation into Escherichia coli inner membrane vesicles could take place under a variety of experimental conditions, with co- or posttranslational assays and proton motive force-dependent or -independent mutants. It was found that translocation can even occur in pure lipid vesicles, under which conditions the tryptophans must directly interact with the lipids. However, under all these conditions tryptophans neither inhibited nor stimulated translocation, demonstrating that they do not affect topology and suggesting that this may be universal for tryptophans in membrane proteins. In contrast, we could demonstrate that lysines clearly prefer to stay on the cis-side of the membrane, in agreement with the positive-inside rule. A statistical analysis focusing on interfacially localized residues showed that in single-spanning membrane proteins lysines are indeed located on the inside, while tryptophans are preferentially localized at the outer interface. Since our experimental results show that the latter is not due to a topology-determining role, we propose instead that tryptophans fulfill a functional role as interfacially anchoring residues on the trans-side of the membrane.

Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain.

Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.

Antibody response in cats to the envelope proteins of feline immunodeficiency virus: identification of an immunodominant neutralization domain.

Overlapping fragments of the envelope protein of feline immunodeficiency virus (FIV) have been expressed in Escherichia coli. Screening of cat sera for antibodies to these fragments revealed that the immunodominant domain of the FIV envelope is localized within the transmembrane protein (amino acids 687-741) and that both the variable region 3 (SU3, aa 385-417) and the COOH-terminus (aa 599-611) of the surface protein (SU) are highly immunogenic. Of all rabbit sera raised to the envelope protein fragments only the serum directed to SU3 was neutralizing. Both FIV-infected and SU3-immunized cats elicited neutralizing antibodies to SU3. Neutralizing antibodies in sera of infected cats could be absorbed by SU3, showing that SU3 is a major neutralization domain of FIV.

Evaluation of subunit vaccines against feline immunodeficiency virus infection.

Subunit vaccines prepared against feline immunodeficiency virus (FIV) infection were evaluated in two trials. First, cats were immunized with bacterial expression products of an envelope fragment that contained the V3 neutralization domain of the FIV surface protein fused to either galactokinase (K-SU3) or glutathione-S-transferase (G-SU3). Quantitative and qualitative differences in the humoral immune response were observed with three adjuvants of which Quil A was the best in terms of total and virus neutralizing antibody. Notwithstanding the responses induced, 19 of 20 immunized cats did not resist challenge and became infected. To determine whether priming with a live viral vector would confer protection, cats were inoculated oronasally and subcutaneously with a feline herpesvirus (FHV) mutant expressing the FIV env gene; two booster immunizations followed using the K-SU3 product in either Quil A or a mineral oill Al(OH)3 adjuvant. FIV-specific antibody responses were only weak, and the vaccinates did not withstand challenge with a low dose of homologous virus.