Apoptosis in the malaria protozoan,Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito

Death by apoptosis regulates cell numbers in metazoan tissues and it is mediated by activation of caspases and results in characteristic morphological and biochemical changes. We report here that the malaria protozoan, Plasmodium berghei, exhibits features typical of metazoan apoptotic cells including condensation of chromatin, fragmentation of the nuclear DNA and movement of phosphatidylserine from the inner to the outer lamellae of the cell membrane. In addition, proteins with caspase-like activity were identified in the cytoplasm of the ookinete suggesting that the cellular mechanism of cell death may be similar to that of multicellular eukaryotes. Our data show that more than 50% of the mosquito midgut stages of the parasite die naturally by apoptosis before gut invasion. Cell death was prevented by a caspase inhibitor, treatment resulting in a doubling of parasite intensity. All these features also occur in vitro. Cell suicide thus plays a major and hitherto unrecognised role in controlling parasite populations and could be a novel target for malaria control strategies.     

Mutations in RIPK4 cause the autosomal-recessive form of popliteal pterygium syndrome

The autosomal-recessive form of popliteal pterygium syndrome, also known as Bartsocas-Papas syndrome, is a rare, but frequently lethal disorder characterized by marked popliteal pterygium associated with multiple congenital malformations. Using Affymetrix 250K SNP array genotyping and homozygosity mapping, we mapped this malformation syndrome to chromosomal region 21q22.3. Direct sequencing of RIPK4 (receptor-interacting serine/threonine kinase protein 4) showed a homozygous transversion (c.362T>A) that causes substitution of a conserved isoleucine with asparagine at amino acid position 121 (p.Ile121Asn) in the serine/threonine kinase domain of the protein. Additional pathogenic mutations-a homozygous transition (c.551C>T) that leads to a missense substitution (p.Thr184Ile) at a conserved position and a homozygous one base-pair insertion mutation (c.777_778insA) predicted to lead to a premature stop codon (p.Arg260ThrfsX14) within the kinase domain-were observed in two families. Molecular modeling of the kinase domain showed that both the Ile121 and Thr184 positions are critical for the protein's stability and kinase activity. Luciferase reporter assays also demonstrated that these mutations are critical for the catalytic activity of RIPK4. RIPK4 mediates activation of the nuclear factor-κB (NF-κB) signaling pathway and is required for keratinocyte differentiation and craniofacial and limb development. The phenotype of Ripk4(-/-) mice is consistent with the human phenotype presented herein. Additionally, the spectrum of malformations observed in the presented families is similar, but less severe than the conserved helix-loop-helix ubiquitous kinase (CHUK)-deficient human fetus phenotype; known as Cocoon syndrome; this similarity indicates that RIPK4 and CHUK might function via closely related pathways to promote keratinocyte differentiation and epithelial growth.     

Naturally Occurring Triggers that Induce Apoptosis-Like Programmed Cell Death in Plasmodium berghei Ookinetes

Several protozoan parasites have been shown to undergo a form of programmed cell death that exhibits morphological features associated with metazoan apoptosis. These include the rodent malaria parasite, Plasmodium berghei. Malaria zygotes develop in the mosquito midgut lumen, forming motile ookinetes. Up to 50% of these exhibit phenotypic markers of apoptosis; as do those grown in culture. We hypothesised that naturally occurring signals induce many ookinetes to undergo apoptosis before midgut traversal. To determine whether nitric oxide and reactive oxygen species act as such triggers, ookinetes were cultured with donors of these molecules. Exposure to the nitric oxide donor SNP induced a significant increase in ookinetes with condensed nuclear chromatin, activated caspase-like molecules and translocation of phosphatidylserine that was dose and time related. Results from an assay that detects the potential-dependent accumulation of aggregates of JC-1 in mitochondria suggested that nitric oxide does not operate via loss of mitochondrial membrane potential. L-DOPA (reactive oxygen species donor) also caused apoptosis in a dose and time dependent manner. Removal of white blood cells significantly decreased ookinetes exhibiting a marker of apoptosis in vitro. Inhibition of the activity of nitric oxide synthase in the mosquito midgut epithelium using L-NAME significantly decreased the proportion of apoptotic ookinetes and increased the number of oocysts that developed. Introduction of a nitric oxide donor into the blood meal had no effect on mosquito longevity but did reduce prevalence and intensity of infection. Thus, nitric oxide and reactive oxygen species are triggers of apoptosis in Plasmodium ookinetes. They occur naturally in the mosquito midgut lumen, sourced from infected blood and mosquito tissue. Up regulation of mosquito nitric oxide synthase activity has potential as a transmission blocking strategy.     

Enhancement of alkaline protease production in Bacillus circulans using plasmid transformation

Plasmid transformation is an efficient and crucial biotechnological tool that enables the enhancement of many important microbial characters that would be beneficial in a lot of industrial, agricultural and environmental applications. In the present study, five Bacillus species (B. subtilis, B. cereus, B. alvei, B. circulans and B. pumilus) were investigated. They were isolated from agricultural soils of different local arid environments of the Kingdom of Saudi Arabia, identified and characterized for their plasmid content. The main objective of the present study was to enhance the production of alkaline protease in Bacillus circulans (the recipient strain) through plasmid transformation from B. subtilis (the donor strain). All the tested Bacillus strains successfully produced unique multiple (3, 4 and 5) spontaneous antibiotic resistant mutants against chloramphenicol, neomycin, rifampicin, streptomycin, kanamycin and tetracycline and all of which were mutated to Rif_r strains. B. pumilus showed the highest resistance against five of the six tested antibiotics while both of B. alvei and B. circulans showed the lowest resistance to only three of the tested antibiotics. Results revealed that B. subtilis was the best among the tested species concerning the production of alkaline protease (90.2 U/ml) while B. pumilus was the lowest in activity (40.3 U/ml). Screening of plasmid content revealed the presence of one or two mega indigenous plasmids in all the tested species. The four transformant strains BC ₁ , BC ₂ , BC ₃ and BC ₄ resulting from plasmid transformation exhibited significant increases in the activity of alkaline protease and recorded 2.31- to 3-fold increases compared to the parent B. circulans cells and 2.11- to 2.75-fold increases compared to the donor cells of B. subtilis. They also acquired antibiotic resistance to tetracycline and chloramphenicol that was completely absent in the parent cells of B. circulans. Results revealed that plasmid transformation among the tested Bacillus spp. is a powerful technique that can be efficiently exploited to enhance alkaline protease production in the transformed Bacillus spp. compared to their wild strains and we recommend using the improved transformant strains for commercial and industrial purposes.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Developmental distribution of the polyadenylation protein CstF-64 and the variant tauCstF-64 in mouse and rat testis

Messenger RNA polyadenylation is one of the processes that control gene expression in all eukaryotic cells and tissues. In mice, two forms of the regulatory polyadenylation protein CstF-64 are found. The gene Cstf2 on the X chromosome encodes this form, and it is expressed in all somatic tissues. The second form, tauCstF-64 (encoded by the autosomal gene Cstf2t), is expressed in a more limited set of tissues and cell types, largely in meiotic and postmeiotic male germ cells and, to a smaller extent, in brain. We report here that whereas CstF-64 and tauCstF-64 expression in rat tissues resembles their expression in mouse tissues, significant differences also are found. First, unlike in mice, in which CstF-64 was expressed in postmeiotic round and elongating spermatids, rat CstF-64 was absent in those cell types. Second, unlike in mice, tauCstF-64 was expressed at significant levels in rat liver. These differences in expression suggest interesting differences in X-chromosomal gene expression between these two rodent species.     

In vitro methods for culturing vertebrate and mosquito stages of Plasmodium

The development of in vitro culture systems for the vertebrate stages of Plasmodium led to major advancements in malaria research. Here we review both improvements made in these techniques and the recent achievement of the in vitro growth of mosquito stages from ookinete to infective sporozoite.     

Optimization and/or acclimatization of activated sludge process under heavy metals stress

The present study aimed to overcome the toxicity of the heavy metals load, discharged with the industrial effluents into Alexandria sewerage network, on the activated sludge treatment system through effective acclimation for organic matter and heavy metals removal. Optimization and/or acclimatization of the activated sludge process in the presence of Cu, Cd, Co and Cr contaminating mixed domestic-industrial wastewater was investigated. Acclimatization process was performed through abrupt and stepwise addition of tested metals using sequencing batch reactors treatment approach and evaluated as microbial oxygen uptake rate (OUR), dehydrogenase activity (DHA), organic matter (COD) and heavy metals removal. Abrupt addition of metals adversely affected sludge bioactivity leading to decline in the removal efficiency of the targeted contaminants and loss of floc structure. Metals IC₅₀ confirmed that copper possessed the highest toxicity towards the OUR, DHA activity and COD removal with orders Cu > Cd > Cr > Co; Cu > Cd > Co = Cr and Cu > Cd > Cr > Co, respectively. The highest metal removal was recorded for Cd followed by Co, Cu and finally Cr, most of which was retained in the dissolved influent. However, controlled stepwise application of the tested metals exhibited high sensitivity of DHA and OUR activities only at the highest metal concentrations although enhanced at the lowest concentrations while COD removal was not significantly affected. In conclusion, this approach resulted in adaptation of the system where sludge microbes acquired and developed natural resistance to such metals leading to remarkable enhancement of both organic matter and heavy metals removal.     

Toxocara Awareness Among Medical Practitioners in Saudi Arabia

Human toxocariasis is a zoonotic infection with global and regional impacts. Worldwide it is underestimated and clinically overlooked. Medical practitioners are generally unaware of the extent of the resulting disease spectrum. The objective of the study was to assess knowledge and disease awareness among medical practitioners in Aseer, south-western Saudi Arabia. A questionnaire addressing knowledge about the parasite, its visceral larva migrans and the disease spectrum generated was used to interview participants. The study included 285 participants. In answer to the question what is toxocara, only 27%, answered correctly that it is a nematode, paediatricians being the majority. With regard to years of experience among participants, 56.8% of those who answered correctly had less than 5-year experience, as opposed to 35.4% for those with more than 10-year experience. The cumulative awareness about the disease manifestations and spectrum, i.e. those who knew, was less than 30% across specialties and years of experiences. Lack of awareness regarding Toxocara infection and the disease spectrum it can generate is evident. The consequence for such lack of knowledge within our practising medical community is simply unacceptable as it might translate into misdiagnosis and consequently misguided treatment.

     

Bioremediation of Vegetable Oil and Grease from Polluted Wastewater Using a Sand Biofilm System

Pseudomonas sp. (L1), P. diminuta(L2) were among eight bacterial strains isolated from vegetable grease and oil-contaminated industrial wastewater, four of which only were found to have the ability to degrade oil and grease. They were identified and investigated for oil and grease degradation either individually or in combinations in previous unpublished work by the authors. Since the combination M1 (Pseudomonas sp. andP. diminuta) produced the highest degradative activity, it was used in the present study in a biofilm sand filter system for vegetable oil and grease removal. This system was tested either as one unit or two units in sequence where different flow rates (30, 50, 100 ml/h) were applied compared to a control unit(s). Results showed that both biofilm systems reduced oily wastewater, even in cases of high degree of pollution (fat, oil & grease (FOG), 7535 ppm; biochemical oxygen demand (BOD₅), 525 ppm; chemical oxygen demand (COD), 1660 ppm). Results also showed a removal of FOG with efficiency at 100%; BOD₅ at 95.9% and COD at 96%, at 50 ml/h flow rate using one unit of biofilm system. On using two units in sequence, a complete removal of FOG, BOD₅ and COD with efficiency 100%, at flow rate 100 ml/h was achieved. In conclusion, the previous biofilm results indicated the efficiency of such a system in treating oily polluted wastewater (vegetable oil origin) on the basis of bacterial isolates being used, the optimum flow rate, and the number of biofilm units used in sequence to obtain the highest removal capacity of such a system. This revised version was published online in July 2006 with corrections to the Cover Date.