Synthesis and biological evaluation of 3-(4-chlorophenyl)-4-substituted pyrazole derivatives.

3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro antifungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core pyrazole scaffold may be promising antifungal and antitubercular agents.

Expression of var genes located within polymorphic subtelomeric domains of Plasmodium falciparum chromosomes.

Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.

A MAP kinase homologue from the human malaria parasite, Plasmodium falciparum.

Pfmap-1, a gene encoding a novel protein kinase, has been identified in the human malaria parasite Plasmodium falciparum, using the polymerase chain reaction with degenerate oligodeoxyribonucleotides designed to hybridise to conserved regions of cdc2-related kinases. Computer comparison with other protein kinases strongly suggests that the protein encoded by this gene is closely related to mitogen-activated protein (MAP) kinases, which play important roles in eukaryotic adaptative response and signal transduction. In addition to the conserved MAP kinase catalytic domain, Pfmap-1 contains a highly charged C-terminal extension that includes two sets of repeated amino acid motifs. Pfmap-1 is located on chromosome 14 of P.falciparum, and its mRNA has a size of 3.7 kb.

A distinct member of the aspartic proteinase gene family from the human malaria parasite Plasmodium falciparum.

A gene (hap) transcribed during the intra-erythrocytic life cycle stages of the human malaria parasite Plasmodium falciparum was cloned and sequenced. It was found to encode a protein belonging to the aspartic proteinase family but which carried replacements of catalytically crucial residues in the hallmark sequences contributing to the active site of this type of proteinase. Consideration is given as to whether this protein is the first known parasite equivalent of the pregnancy-associated glycoproteins that have been documented in ungulate mammals. Alternatively, it may be operative as a new type of proteinase with a distinct catalytic mechanism. In this event, since no counterpart is known to exist in humans, it affords an attractive potential target against which to develop new anti-malarial drugs.

Assessing the impact of Plasmodium falciparum genome sequencing.

With the publication of the complete sequences for chromosomes 2 and 3 and the increasing availability of shotgun sequence covering most of its genome, Plasmodium falciparum biology is entering its post-genomic era. Analysis of the results generated to date has identified higher-order organisation of gene families involved in parasite pathology, provided information regarding the unique biology of this parasite and allowed the identification of potential chemotherapeutic drug targets. Continuing efforts to complete the P. falciparum genome and the availability of sequences from other protozoan parasites will facilitate a broader understanding of their biology, particularly with respect to their pathogenicity.

Mutational analysis identifies a five base pair cis-acting sequence essential for GBP130 promoter activity in Plasmodium falciparum.

Here we describe the functional characterization of a Plasmodium falciparum promoter region, identifying a discrete five base pair sequence element that is responsible for efficient promoter activity. This sequence element binds nuclear factors in a sequence-specific manner. It shares no homology with any known eukaryotic transcription factor binding site, supporting the notion that the protozoan parasite P. falciparum has evolved a transcriptional machinery distinct from that of its human and mosquito hosts. This report represents the first description of a minimal and necessary cis-acting sequence element for efficient promoter activity in P. falciparum.

Control of gene expression in Plasmodium falciparum.

Transfection has facilitated a functional analysis of transcriptional processes in the human malarial parasite Plasmodium falciparum, providing the first fascinating glimpses into the mechanisms regulating parasite development and pathogenicity. Here we review our rapidly evolving knowledge of what constitutes a promoter, what factors regulate promoter activity and how this activity affects the manifestation of the disease.

Physical and functional mapping of the transcriptional start sites of Plasmodium falciparum proliferating cell nuclear antigen.

RNase protection assays and primer extension analysis have been used to locate a major transcription start site 960 bp upstream from the translational start of the PfPCNA coding sequence. A second, minor, site is situated a further 40 bp upstream. Intraerythrocytic parasite stages were transiently transfected with constructs containing a firefly luciferase reporter gene under the transcriptional control of variously modified elements of the PfPCNA 5' flanking sequence. These experiments identified a 470 bp region essential for promoter activity, which contains the physically mapped transcriptional start sites. In addition, a region between 290 and 620 bp upstream of the transcriptional start sites is required for efficient promoter activity.

Intraerythrocytic polyubiquitin expression in Plasmodium falciparum is subjected to developmental and heat-shock control.

The polyubiquitin gene of the human protozoan parasite Plasmodium falciparum (PfpUB) was cloned and shown to be comprised of five tandem repeats of the ubiquitin open reading frame, present as a single copy on chromosome 12. The 1672 bp of PfpUB is interrupted at the 5' end by a single intron of 526 bp. PfpUB expression is developmentally regulated in intraerythrocytic stages with a marked increase in both steady-state transcript and polyubiquitin protein levels in late trophozoite stages. On response to heat shock, late stage parasites (late trophozoites and schizonts) have a slightly elevated PfpUB transcript level as well as readily observable increases in the amount of polyubiquitin and ubiquitin-conjugated proteins.

Intraerythrocytic expression of topoisomerase II from Plasmodium falciparum is developmentally regulated.

The stage-specific relationship between promoter activity, transcript production, protein expression and enzyme activity has been investigated for the gene encoding Plasmodium falciparum topoisomerase II (PfTopoII). Nuclear run-on experiments have shown that the P. falciparum topoisomerase II gene (PfTOP2) promoter is active at low levels in ring stage parasites, but reaches high levels of activity as the parasites progress into trophozoite/schizont asexual stages. Steady-state PfTOP2 transcripts are present at low levels in rings, accumulate in trophozoites, but are completely undetectable in schizonts. An antiserum raised against the species-divergent carboxy-terminus of PfTopoII, which neutralised the decatenation activity in parasite extracts, was used to probe Western blots of ring, trophozoite and schizont stage parasite extracts. Relatively low levels of PfTopoII were seen in rings compared with those in trophozoite and schizont preparations. Parasite extracts were also used to compare the patterns of protein accumulation and enzyme activity at these stages. Complete decatenation of kinetoplast substrate DNA (KDNA) was found in schizont stages, very low levels of activity were observed in rings and trophozoites showed intermediate levels. These finding show that, as parasites progress towards the stages where DNA replication occurs, there is a concomitant increase in both topoisomerase II production and activity.

Pfcrk-1, a developmentally regulated cdc2-related protein kinase of Plasmodium falciparum.

A gene encoding a novel cdc2-related protein kinase has been identified in Plasmodium falciparum, using degenerate oligonucleotides designed to hybridise to regions that are conserved in members of the cdc2 gene family. This gene, called Pfcrk-1, is located on chromosome 4. It is most closely related to the p58GTA gene family, members of which are negative regulators of cell growth in vertebrates. Pfcrk-1 is developmentally regulated, as indicated by stage-specific accumulation of mRNA in gametocytes.

Stage specific expression of proliferating cell nuclear antigen and DNA polymerase delta from Plasmodium falciparum.

Antisera raised against proliferating cell nuclear antigen (PfPCNA) and DNA polymerase delta (PfDNA Pol delta) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfDNA Pol delta also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfDNA Pol delta promoter activity is absent in rings but present in trophozoites and schizonts, the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication.

Zahn's 'infarcts' of the liver.

Three patients were found at necropsy to have Zahn's ;infarcts' of the liver. In one of these cases the ;infarct' merely showed severe centrilobular congestion. In the other two cases there was centrilobular necrosis, and in one of these early fibrosis was seen. Portal vein occlusion was present in all three cases and this had followed splenectomy in two of them. There was no evidence of hepatic artery or vein occlusion. Circulatory failure is considered to have played a part in the pathogenesis of the lesions.

Qualitative and quantitative abnormalities of von Willebrand antigen in patients with diabetes mellitus.

Previous reported studies of vVWf antigen (vWf:Ag) in patients with diabetes mellitus have not shown qualitative abnormalities despite frequent documentation of raised vWf:Ag. Twenty two patients with established diabetes mellitus have been studied and qualitative abnormalities of vWf:Ag multimers were found in 10 patients who had poorer glycaemic control (as judged by plasma fructosamine) than the other 12 patients. Seven of the 22 patients were restudied after a 3 month period of improved glycaemic control and the vWf:Ag multimer abnormalities disappeared in 6 of these. Some of the structural abnormalities were accompanied by loss of vWf functional activity. As vWf:Ag is synthesised in the endothelial cell, vWf:Ag abnormalities during periods of poor glycaemic control may reflect endothelial cell damage with vWf:Ag release and possibly subsequent proteolysis.

Urinary pregnanetriol excretion in hirsutism.

Eighteen consecutive hirsute women attending an endocrine clinic have been studied by measurement of the urinary pregnanetriol excretion before and following the concurrent administration of corticotrophin and metyrapone. An abnormal increment in pregnanetriol excretion was observed in 11 of these 18 patients. It is suggested that this is evidence that an adrenal abnormality, probably operative at the 21-hydroxylase level, might be a factor responsible for the hirsutism in these 11 patients. Five adrenalectomized women who were also studied showed no significant increase in urinary pregnanetriol excretion in response to concurrent corticotrophin and metyrapone administration.

Differences in nucleosome organization over episomally located plasmids coincides with aberrant promoter activity in P. falciparum.

Here we investigated whether the Plasmodium falciparum GBP130 promoter maintains its developmental activity during the intraerythrocytic cycle when located on an episomal plasmid introduced using transient transfection. Comparing its activity with that of the endogenous chromosomally located GBP130 promoter indicates that the episomally located GBP130 promoter looses its developmental restriction, being rendered constitutively active. Loss of developmental restriction coincides with the absence of phased nucleosomal arrays over the episome. These data suggest that epigenetic factors may play a role in developmentally regulated gene expression in P. falciparum.

A self-help group for complete denture wearers.

Denture provision is a frequent source of frustration and misunderstanding for both patients and dentists. This study investigates the role of a self-help group in helping denture wearers with long standing problems to come to terms with their difficulties and to reduce their demands on the dentist. Eleven people with clinically satisfactory dentures and long standing perceived problems were invited to join the programme. Evaluation at 2 and 12 months afterwards indicated everyone had benefited in some way. Benefits included eating foods previously avoided, the opportunity for social intercourse and reducing the visits to the dentist.