Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy

It has been shown that adoptive immunotherapy can be curative for established malignant tumors. The key to this treatment lies in obtaining sufficient numbers of lymphocytes which are sensitized to recognize tumor antigens and carry out immunological reactions to destroy tumor cells. Reported here are the results of experiments to: 1) sensitize lymphocytes to the antigens of rat glioma cells and expand them ex vivo for use in adoptive immunotherapy, 2) characterize the cells of the expanded population, and 3) evaluate antitumor activity in a cohort of rats with well-established intracranial gliomas. Viable RT-2 glioma cells were injected into the hind foot pads of syngeneic Fischer 344 rats. After 10 days, the tumor draining lymph nodes (DLN) were harvested from the injected limbs and mechanically dissociated. The cells of the DLN were then suspended in culture medium supplemented with low dose interleukin-2 (IL-2) and incubated for 18 hours with Bryostatin-1 and ionomycin (Bryo/Io) to stimulate expansion. The cells were next washed to remove the Bryo/Io and resuspended in culture medium and IL-2. Population expansions of 40- to 100-fold were seen after 8 days. Flow cytometric analysis showed these cells to be a nearly pure population of T lymphocytes of the CD3+CD8+ phenotype. Intravenous injection of the ex vivo expanded DLN cells did not significantly improve survival of rats with a seven-day intracerebral RT-2 glioma, although, compared to untreated controls, the tumors of the treated animals were smaller, showed no necrosis, and appeared to be less infiltrative. Furthermore, the treated animals had a pronounced lymphocytic infiltration of their tumors with greater associated degrees of hemorrhagic change and peritumoral edema. When the ex vivo expanded DLN cells were intravenously injected into three-day intracerebral RT-2 glioma models, tumors were almost always eliminated and the animals survived their tumor challenge. We conclude that successful expansion of glioma-sensitized DLN lymphocytes is possible and that adoptive immunotherapy using these cells is capable of effectively limiting the progression of large gliomas, while totally eradicating small ones.     

Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy

Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA.     

Structural organization of the intact bacterial cellulosome as revealed by electron microscopy

The architecture of the intact cellulosome of Clostridium thermocellum, a huge extracellular multi-polypetide bacterial enzyme complex engaged in degradation of cellulose, was investigated by electron microscopy. This was done because former electron microscopic studies aimed at elucidation of the structure of polycellulosomes and cellulosomes were restricted by the fact that data on macromolecular details could only be derived from deformed or disrupted enzyme complexes, or by application of cryo preparation and imaging techniques yielding insufficient resolution. The shape of well-preserved cellulosomes was more or less spherical, often similar to that of an olive fruit with a cavity. Therein, multiple fibrillar structures could be visualized, interpreted to be the proximal stretches of copies of the fibrillar protein Cip A ('scaffoldin'), the nonenzymatic scaffolding protein known to function as attachment site for the enzymatic subunits, as well as fibrillar parts of anchoring proteins. The enzymatic subunits were depicted to be attached, in a repetitive fashion, to the distal stretches of the Cip A proteins. The enzymatic subunits were seen, in the intact cellulosome, to form a shell-like complex substructure surrounding the cavity. Obviously, this kind of architecture makes sure that the catalytic domains of the enzymatic subunits are exposed to the environment, and, hence, to the substrate, the cellulose fibrils. Attempts were made to demonstrate the alternating occurrence of coiled domains and fibrillar stretches along the elongated protein Cip A previously characterized by sequencing, X-ray, and NMR studies. To this end, Cip A molecules, with adhering enzymatic subunits, were partially removed from their native location within the cellulosome, "stretched" by hydromechanical forces directly on the electron microscopic support film, negatively stained, and depicted by electron microscopy. The alternating occurrence of presumed coiled domains and fibrillar stretches along Cip A could be visualized, together with detached enzymatic subunits found on the support film.     

Quasi- and nonequivalence in the structure of bacterial flagellar filament

In supercoiled forms of flagellar filaments, which are thought to be produced by combinations of two distinct subunit lattices, the lattices are elastically deformed in 11 different ways, depending on their azimuthal positions on the circumference of a tube with 11 protofilaments. Those two interactions are nonequivalent as opposed to quasiequivalent ones in elastically deformed lattices of otherwise identical interactions. The term nonequivalence is defined to represent different bonding interactions, and quasiequivalent is used to describe deformed but conserved bonding interactions. By using two distinct lattices that were accurately determined by x-ray fiber diffraction, 10 possible supercoiled forms of flagellar filaments were simulated, based on a bistable-subunit packing model. Comparison to the observed forms showed good agreement, indicating that the model and determined lattice parameters effectively represent realistic features of the structure. The simulated quasiequivalent lattices have been compared to the two nonequivalent lattices, revealing an interesting feature: the maximum deviation in the intersubunit distance by elastic deformation is almost three-quarters of the difference between the two distinct lattices, demonstrating a balanced coexistence of a well-defined conformational distinction and extensive adaptability in the molecular structure of flagellin and its packing interactions.     

Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene

Leukotrienes constitute a class of potent biological mediators of inflammation and anaphylaxis (for reviews see refs 1 and 2). Their biosynthesis derives from 5-lipoxygenase-catalysed oxygenation of arachidonic acid in granulocytes, macrophages and mast cells. To examine the physiological importance of leukotrienes, we have disrupted the 5-lipoxygenase gene by homologous recombination in embryonic stem cells. 5-Lipoxygenase-deficient (5LX-/-) mice develop normally and are healthy. They show a selective opposition to certain inflammatory insults. Although there is no difference in their reaction to endotoxin shock, the 5LX-/- animals resist the lethal effects of shock induced by platelet-activating factor. Reaction to ear inflammation induced by phorbol ester is normal, whereas inflammation induced by arachidonic acid is markedly reduced. Contrasts were also found in two models of leukocyte chemotaxis in vivo. The phenotype of 5LX-/- mice under injurious insult identifies the role for leukotrienes in the pathophysiology of select inflammatory states.     

An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.     

Role of lipopolysaccharide in colonization of the mouse intestine by Salmonella typhimurium studied by in situ hybridization

An avirulent, streptomycin-resistant Salmonella typhimurium strain, SL5319, and its lipopolysaccharide (LPS)-deficient mutant strain, SL5325, differ in their ability to colonize the large intestines of streptomycin-treated mice. When fed to mice independently, the strains colonize equally well, but when fed together, the LPS-deficient mutant is outcompeted by the wild-type strain during establishment in the gut (J.J. Nevola, B.A.D. Stocker, D.C. Laux, and P.S. Cohen, Infect. Immun. 50:152-159, 1985). In the present study, the spatial distribution in the intestinal mucosal layer of the two strains was visualized by specific hybridization to bacterial rRNA in histological sections of mouse colon and cecum. The first day after infection, 9.8% of the smooth SL5319 cells observed in mucus were found to be associated with the mouse epithelial cells, but three days after infection, the corresponding fraction of adhering bacteria was reduced to 2.1%. The LPS-deficient S. typhimurium strain was confined to the part of the mucosal layer closest to the colonic lumen and was not observed to adhere to the epithelium either at day 1 or 3 after infection. Quantitative determinations of the distance from the S. typhimurium cells to the epithelial wall confirmed that the average distance for the rough S. typhimurium SL5325 was much larger than for its smooth counterpart, S. typhimurium SL5319. Quantification of the hybridization signal from bacteria isolated from the cecal mucus revealed that the two strains had the same ribosome concentration, indicating that they have the same potential for growth in the intestinal environment. On the basis of these observations, we suggest that the better colonization ability of the strain carrying wild-type LPS is due to the better abilities to penetrate the intestinal mucosal layer and to subsequently bind to the epithelial cells in vivo.     

Differences in black pigmentation in lepidopteran cuticles as revealed by light and electron microscopy

Black cuticles of larvae and pupae from various Lepidoptera were studied by light and electron microscopy. There are striking differences in the representation of black pigmentation, especially at the ultrastructural level. Two types may be described: 1. With the light microscope black melanin-like grana, electron-dense electron microscopically, are found in the distal parts of the exocuticle. This type is demonstrated in larvae of Celerio euphorbiae, Papilio machaon, and Phalera bucephala. 2. With the light microscope, a dark homogeneous layer in the distal exocuticle can be recognized, however, electron microscopically no structures correlated with this dark pigment layer. This type of pigmentation was present in pupae of Pieris brassicae and Aglais urticae; in Pieris larvae the dark pigmented layer appeared to be limited to the epicuticle. In Celerio processes of the epidermal cells are involved in transporting precursors to the exocuticle. The conclusion was reached that black pigmentation in cuticles is based on different mechanisms as proposed by structural features. The two likely mechanisms are melanization and sclerotization.     

Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99. Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.     

Lipid domain structure of the plasma membrane revealed by patching of membrane components

Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.     

The sporulation process in Thermomonospora fusca as revealed by scanning and transmission electron microscopy

The sporulation process in the thermophilic actinomycete Thermomonospora fusca was observed by scanning and transmission electron microscopy. As shown by scanning electron microscopy, spores were produced primarily on aerial hyphae and first appeared as bud-like enlargements at the tips of short multibranched sporophores. Young spores were oval to spherical in shape with a smooth surface. As they matured spores enlarged and developed a rough and globular covering, which was quite fragile and easily detached from the spore. This outer layer, as observed by transmission electron microscopy, was thought equivalent to the sheath of other Thermomonospora species. In cross section, mature spores exhibited a thick spore coat underneath the outer globular layer. This spore coat was usually observed as a single layer, but some spores produced a bilayered coat. No multilayered spore coat or spore cortex was observed in the heat-sensitive spores of T. fusca. They were, therefore, shown to be aleuriospores (microcondia), and not endospores.     

Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 A resolution

Using a liquid-helium-cooled superconducting electron cryo-microscope, we obtained low-dose images of negatively stained preparations at 4 K and collected structural data to 1/9.6 -1 for flagellar filaments from the strain SJW117 of Salmonella typhimurium (serotype gt). The subunits of this left-handed, straight filament are non-helically perturbed in a pairwise manner. The perturbation corresponds to an alternating conformation in every other row of subunits. These are the 5-start rows and, necessarily, the resulting structure has a seam. The perturbation is not confined to the outside but extends into the structure. We separated the non-symmetric and symmetric parts of the structural data and generated a three-dimensional reconstruction from the latter. The resulting density map is a structure similar in domain organization to the left-handed filament of S. typhimurium SJW1660. Filtered images generated from the non-symmetric component show an ordered and polar structure. The nature of the perturbation was analyzed by model building using a sphere to represent the subunit at low resolution. A lateral shift of approximately 10 degrees mimics the perturbation.     

Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium

Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.     

Role for the Salmonella flavohemoglobin in protection from nitric oxide

Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms. Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus. Determination of a function for these flavohemoglobins has been elusive. A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide. The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function. These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.     

European Y-chromosomal lineages in Polynesians: a contrast to the population structure revealed by mtDNA

We have used Y-chromosomal polymorphisms to trace paternal lineages in Polynesians by use of samples previously typed for mtDNA variants. A genealogical approach utilizing hierarchical analysis of eight rare-event biallelic polymorphisms, seven microsatellite loci, and internal structural analysis of the hypervariable minisatellite, MSY1, has been used to define three major paternal-lineage clusters in Polynesians. Two of these clusters, both defined by novel MSY1 modular structures and representing 55% of the Polynesians studied, are also found in coastal Papua New Guinea. Reduced Polynesian diversity, relative to that in Melanesians, is illustrated by the presence of several examples of identical MSY1 codes and microsatellite haplotypes within these lineage clusters in Polynesians. The complete lack of Y chromosomes having the M4 base substitution in Polynesians, despite their prevalence (64%) in Melanesians, may also be a result of the multiple bottleneck events during the colonization of this region of the world. The origin of the M4 mutation has been dated by use of two independent methods based on microsatellite-haplotype and minisatellite-code diversity. Because of the wide confidence limits on the mutation rates of these loci, the M4 mutation cannot be conclusively dated relative to the colonization of Polynesia, 3,000 years ago. The other major lineage cluster found in Polynesians, defined by a base substitution at the 92R7 locus, represents 27% of the Polynesians studied and, most probably, originates in Europe. This is the first Y-chromosomal evidence of major European admixture with indigenous Polynesian populations and contrasts sharply with the picture given by mtDNA evidence.     

Social structure of the mound-building mouse Mus spicilegus revealed by genetic analysis with microsatellites

The Mound-building mouse Mus spicilegus possesses a unique behaviour amongst mice. It constructs large earthen mounds and associated nesting chambers which serve to store food for immature individuals during the winter nesting period. We have used genetic analysis of four autosomal and four X-linked microsatellite loci to determine relationships between individuals inhabiting 40 mounds in Bulgaria. We show that, in almost all cases, individuals in a mound are the product of multiple parentage. We estimate the minimum number of males and female parents contributing offspring to each mound and demonstrate that at least two male and two female parents contribute offspring to a minimum of seven mounds. Analyses of relatedness coefficients and allele sharing values demonstrate that parents of different sibships within mounds are more related than if they had been chosen at random from the population and suggest that it is the female parents that contribute this excess relatedness. These results suggest that the mechanism by which individuals congregate to build mounds is kin-based and that the evolution of mound building and communal nesting in M. spicilegus is due in part to kin selection. This study represents a novel approach to the study of mammalian behavioural ecology. We have used a genetic dataset to construct an outline of social structure in the absence of behavioural data. These inferences can now be used to direct further work on this species.     

A novel calcium-sensitive switch revealed by the structure of human S100B in the calcium-bound form

Loss of neurons has been considered to be a prime cause of nervous disturbances that occur with advancing age. However, the notion of a constitutive aging-related loss of neurons has been challenged recently in several studies that used up-to-date methods for counting neurons. In this study, we have applied stereological techniques with the objective of obtaining quantitative data on total neuron numbers and the distribution of neuron cross-sectional areas in the fifth cervical (C5) and fourth lumbar (L4) dorsal root ganglion (DRG) of 3- and 30-month-old Sprague-Dawley rats. Tissue data were recorded on a confocal laser-scanning microscope with the use of the optical-disector technique and random, systematic sampling. Aged rats of both sexes disclosed only a small decrease (approximately 12%) in the number of cervical and lumbar DRG neurons. Furthermore, there was no significant correlation between the degree of neuron loss and the extent of behavioral deficits among the aged individuals. The DRG neurons of aged rats had a smaller mean cross-sectional area (approximately 15%; P < 0.001) at both DRG levels. Further analysis of the male cohorts was carried out by using isolectin B4 and neurofilament subunit (phosphorylated 200 kDa; RT97) immunoreactivity (IR) as selective markers for unmyelinated and myelinated axons, respectively, and disclosed no significant change in the relative frequencies of immunoreactive neuron profiles in the old rats. However, RT97-IR DRG neurons of the aged rats had significantly smaller cross-sectional areas (approximately 9% in C5; approximately 16% in L4; P < 0.001) than the young adult rats, indicating a selective cell body atrophy among myelinated primary afferents during aging. The results indicate that loss of primary sensory neurons cannot exclusively explain the functional deficits in sensory perception among senescent individuals. It seems likely that other factors at the subcellular level and/or target interaction(s) contribute substantially to the sensory impairments observed with advancing age.     

Role of decay-accelerating factor in regulating complement activation on the erythrocyte surface as revealed by gene targeting

Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that inhibits both the classical and the alternative pathways of complement activation. DAF has been studied extensively in humans under two clinical settings: when absent from the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) patients, who suffer from complement-mediated hemolytic anemia, and in transgenic pigs expressing human DAF, which have been developed to help overcome complement-mediated hyperacute rejection in xenotransplantation. Nevertheless, the exact role of DAF in regulating complement activation in vivo on the cell surface and the species specificity of this molecule remain to be fully characterized. To address these issues, we have used gene targeting to produce mice lacking GPI-anchored DAF. We found that erythrocytes from mice deficient in GPI-anchored DAF showed no increase in spontaneous complement activation in vivo but exhibited impaired regulation of zymosan-initiated bystander and antibody-triggered classical pathway complement activation in vitro, resulting in enhanced complement deposition. Despite a high level of C3 fixation, no homologous hemolysis occurred. It is noteworthy that GPI-linked DAF knockout erythrocytes, when tested with human and guinea pig sera, were more susceptible to heterologous complement lysis than were normal erythrocytes. These results suggest that DAF is capable of regulating homologous as well as heterologous complement activation via the alternative or the classical pathway. They also indicate that DAF deficiency alone is not sufficient to cause homologous hemolysis. In contrast, when the assembly of the membrane-attack complex is not properly regulated, as in the case of heterologous complement activation or in PNH patients, impaired erythrocyte DAF activity and enhanced C3 deposition could lead to increased hemolytic reaction.