Effective use of gamma irradiation for pathogen inactivation of monoclonal antibody preparations.

Gamma irradiation has been used for decades as an effective method of pathogen inactivation of relatively inert materials. Until recently, its application to biologicals has resulted in unacceptable losses in functional activity. In this report we demonstrate that the damaging secondary effects of gamma irradiation can be controlled while maintaining the pathogen inactivation properties due to damage by primary effects. Control is achieved by a combination of protection from free radical damage to a monoclonal antibody through the use of the antioxidant ascorbate and by freeze-drying to minimize the potential for generating free radicals. The data demonstrate a synergy of these two approaches that results in quantitative recovery of functional activity while maintaining the ability to inactivate greater than 5 logs of porcine parvovirus infectivity.     

Advances of radiation sterilisation in tissue banking

Under the auspices of the IAEA tissue banking programme on “Radiation Sterilisation of Tissue Graft” conducted from 1985 to 2004, many scientists and surgeons were involved in various regional research and development (R&D) projects mainly in dealing with radiation dose selection, radiation effects on human tissues and quality system in radiation sterilisation. New findings on radiation effects, tissue processing and preservation were shared during the regional and interregional meetings and workshops. Many tissue banks started to use radiation (25 kGy) to sterilize tissue grafts for tissue safety and efficacy and still continue to use it. The IAEA Code of Practice for Radiation Sterilization of Tissues Allografts developed in 2007 offered simpler methods to conduct radiation dose setting and dose validation experiments for tissue grafts. Advances in dose selection and dose mapping are continued under the quality management system when banks need to be certified to continue their operation. The combination of good tissue processing and preservation as well as good radiation practice will ensure the tissue products are properly sterilised thus safe and of high quality. Experience in meeting challenges in using radiation sterilisation and achievements reported by the tissue bankers are shared here.     

DNA replication in the malaria parasite

Malaria is increasing as a global problem. Many of the drugs that were effective earlier in this century are now becoming obsolete as the parasite develops resistance to them and, despite earlier hopes, an affordable and effective vaccine remains elusive. It is hoped that a deeper understanding of the parasite's cell and molecular biology will give us a resource for the future and help us to achieve effective control. One aspect of parasite metabolism that has been the subject of recent studies is DNA replication: its timing during parasite development, the enzymes involved and the genes encoding them. In this review John White and Brian Kilbey report on the present status of these studies.     

Effects of gamma irradiation on chromatin activity of sugar beet tissue

Chromatin-associated RNA polymerase activity increases during washing of sugar beet tissue to a maximum by 20 hours. This increase was inhibited by dosages of gamma irradiation between 50 and 400 krad. Template availability, as measured by saturating levels of added Escherichia coli RNA polymerase, also increased with washing and was inhibited, although to a lesser extent, by the above irradiation dosages. Neither endogenous polymerase activity nor template availability was affected by high dosages (300 krad) in unwashed tissue. Exposure of tissue to irradiation (300 krad) at different times during a 20-hour washing period severely inhibited the development of RNA polymerase activity during the early stages of washing. The inhibition of template availability, however, was independent of time of irradiation. The data presented are discussed in relation to the mechanisms involved in the inhibitory effects of gamma irradiation on RNA production and subsequent protein synthesis.     

Actin regulation in the malaria parasite

Many intracellular pathogens hijack host cell actin or its regulators for cell-to-cell spreading. In marked contrast, apicomplexan parasites, obligate intracellular, single cell eukaryotes that are phylogenetically older than the last common ancestor of animals and plants, employ their own actin cytoskeleton for active motility through tissues and invasion of host cells. A hallmark of actin-based motility of the malaria parasite is a minimal set of proteins that potentially regulate microfilament dynamics. An intriguing feature of the Plasmodium motor machinery is the virtual absence of elongated filamentous actin in vivo. Despite this unusual actin regulation sporozoites, the transmission stages that are injected into the mammalian host by Anopheles mosquitoes, display fast (1-3 μm/s) extracellular motility. Experimental genetics and analysis of recombinant proteins have recently contributed to clarify some of the cellular roles of apicomplexan actin monomer- and filament-binding proteins in parasite life cycle progression. These studies established that the malaria parasite employs multiple proteins that bind actin to form pools of readily polymerizable monomers, a prerequisite for fast formation of actin polymers. The motile extracellular stages of Plasmodium parasites are an excellent in vivo model system for functional characterization of actin regulation in single cell eukaryotes.     

Subtilisin-like proteases of the malaria parasite

Proteases play critical roles in the life cycle of the malaria parasite, Plasmodium spp. Within the asexual erythrocytic cycle, responsible for the clinical manifestations of malaria, substantial interest has focused on the role of parasite serine proteases as a result of indications that they are involved in red blood cell invasion. Over the past 6 years, three Plasmodium genes encoding serine proteases of the subtilisin-like clan, or subtilases, have been identified. All are expressed in the asexual blood stages and, in at least two cases, the gene products localize to secretory organelles of the invasive merozoite. They may have potential as novel drug targets. Here, we review progress in our understanding of the maturation, specificity, structure and function of these Plasmodium subtilases.     

The genome of the malaria parasite

The genome of the human malaria parasite Plasmodium falciparum is being sequenced by an international consortium. Two of the parasite's 14 chromosomes have been completed and several other chromosomes are nearly finished. Even at this early stage of the project, analysis of the genome sequence has provided promising new leads for drug and vaccine development.     

The transportome of the malaria parasite

Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.     

Adhesive proteins of the malaria parasite

Malaria infection of the host cells requires host-parasite recognition events mediated by adhesion and signaling molecules. Recent development of systems for stable transformation and targeted integration of exogenous DNA in malaria parasites provides a powerful tool to study the structure and function of Plasmodium attachment motifs, and their role in infection and disease.     

Protein glycosylation in the malaria parasite.

The nature and extent of glycosylation in Plasmodium falciparum has long been controversial. It has been widely believed that O-glycosylation is the major carbohydrate modification in the intraerythrocytic stage of P. falciparum and that the parasite has no N-glycosylation capacity. Contrary to this, recent studies have demonstrated that P. falciparum has a low N-glycosylation capability, and O-glycosylation is either absent or present at an extremely low level, whereas glycosylphosphatidylinositol (GPI) anchor modification is common and is the major carbohydrate modification in parasite proteins. The GPI anchor moieties are essential for parasite survival. The parasite GPI anchors can activate signaling pathways in host cells, and thereby induce the expression of inflammatory cytokines, adhesion molecules and induced nitric oxide synthase (iNOS). This might cause erythrocyte sequestration, hypoglycemia, triglyceride lipogenesis and immune dysregulation. Thus, the parasite GPI anchor structure and biosynthetic pathways are attractive targets for antimalarial and/or antiparasite drug development, as discussed here by Channe Gowda and Eugene Davidson.     

Cerebral malaria pathogenesis: revisiting parasite and host contributions

Cerebral malaria is one of a number of clinical syndromes associated with infection by human malaria parasites of the genus Plasmodium. The etiology of cerebral malaria derives from sequestration of parasitized red cells in brain microvasculature and is thought to be enhanced by the proinflammatory status of the host and virulence characteristics of the infecting parasite variant. In this article we examine the range of factors thought to influence the development of Plasmodium falciparum cerebral malaria in humans and review the evidence to support their role.     

Calcium and the malaria parasite: parasite maturation and the loss of red cell deformability

In the studies reported here, we examined the role of calcium in the maturation of the human malaria parasite Plasmodium falciparum, and in the loss of red cell deformability associated with parasite maturation. P. falciparum alters the permeability of its host red cell, which normally maintains submicromolar cytoplasmic concentrations of calcium. Infection of the red cell and parasite maturation produce a 30-fold increase in calcium uptake. Both parasite maturation and the loss of red cell deformability are blocked by EGTA (by extracellular-free calcium concentrations less than or equal to 35 microM) and by other calcium antagonists. The loss of red cell deformability that occurs with parasite maturation is accompanied by alterations in the cytoskeletal proteins of parasitized red cells similar to those produced by the calcium ionophore A23187 (reductions in bands 2.1 [ankyrin], 4.1, and 5 [actin]). These results establish that parasite development and the loss of red cell deformability are calcium-dependent. They suggest that parasite-induced changes in the calcium permeability of the red cell activate endogenous transglutaminase activity by raising the free calcium concentration of the red cell cytoplasm.