Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.     

Intracellular distribution of iron (and associated elements) in various cell types of larvae and juveniles of the sea lamprey (Petromyzon marinus)

The intracellular distribution of iron and other elements was examined in various cell types in larvae and juveniles of the sea lamprey (Petromyzon marinus) using transmission electron microscopy and energy dispersive x-ray microanalysis. The objective was to establish whether there are cell-type specific relationships between iron and other elements in the iron-rich organs and tissues (adipose tissue, opisthonephric kidneys, dorsal integument, fat column, liver, and posterior intestine) of these two life cycle periods. Iron was localized within either dense bodies (presumptive lysosomes, siderosomes) or in the cytoplasmic matrix of many cell types where it was viewed as haemosiderin/ferritin and ferritin, respectively. Presumptive lysosomes of adipocytes of the nephric folds, dorsal integument, and fat column possessed iron and sulphur and this elemental association was also prevalent in the epithelia of the larval proximal tubules and in the posterior intestine and epidermis of both life periods. Macrophages of the larval haemopoietic tissue (posterior intestine) and of the juvenile opisthonephros, which were described as melanomacrophages because of their granules, possessed iron, sulphur, and calcium. This elemental association was also noted in the presumptive lysosomes of the iron-loaded hepatocytes of the juvenile liver while no elements could be detected in these cells in the larval organ. The variations and similarities in elemental associations between the cell types in each life period and at different life periods is discussed in the context of specific cell functions related to the prevention of iron toxicity. These functions are sequestration of iron and storage as the less toxic haemosiderin (larval adipocytes, macrophages, juvenile hepatocytes) or as part of a process of elimination of excesses of this metal (posterior intestine, dorsal epidermal cells). Due to its unique ability to deal with copious amounts of iron at all periods of the life cycle, the lamprey serves as an important model for studies of iron loading in vertebrates.     

The kidney,adrenocortical homolog,and corpuscles of Stannius in the cockscomb pricklebackAnoplarchus purpurescens

The morphology of the kidney, adrenocortical homolog, and the corpuscles of Stannius was examined in the cockscomb prickleback,Anoplarchus purpurescens, a marine teleost which inhabits the intertidal zone. The paired kidneys of this fish are fused throughout most of their length, there is essentially a single posterior cardinal vein on the right side, they possess renal corpuscles, and there is no distal segment of the tubule. The tubule is specialized, in descending order, into ciliated neck and two proximal segments before entering the system of collecting tubules and ducts. The cells of the latter system are specialized for mucous secretion, as are cells of the main excretory ducts, the paired archinephric ducts. Tubulogenesis occurs in the kidneys in close apposition to the archinephric ducts. The presumptive adrenocortical homolog is located around the posterior cardinal veins in the head kidney while paired corpuscles of Stannius are confined to the posterior end of the kidney. All of the above features are consistent with those found in the kidneys of many other marine teleosts.     

Extensive variation in the O-antigen gene cluster within one Salmonella enterica serogroup reveals an unexpected complex history

The 46 serogroups of Salmonella enterica have different O-antigens, and each is thought to have a specific form of the O-antigen cluster. Comparison of the 145 serovars of serogroup B revealed much more intraserogroup genetic diversity than expected. The O27 factor, due to an alpha 1-6 linkage between O units in place of the more common alpha 1-2 linkage and previously thought to be due to a converting bacteriophage, is now shown to be due to a wzy(alpha(1-6)) gene located within the major gene cluster. Surprisingly a remnant of this gene in all O27(-) serovars shows that the ancestor was O27(+). There are six distinct gene cluster forms, five apparently derived by a series of deletions and one by an insertion from an ancestral O27(+) form present in 57 serovars. The history of the gene cluster and movement between subspecies I and II can be traced. Two of the derivative forms still have a functional wzy(alpha(1-6)) gene, while in three it has been inactivated by deletion or insertion. Two of the forms lacking a functional wzy(alpha(1-6)) gene have the wzy(alpha(1-2)) gene first described for strain LT2 as rfc, whereas for the third the wzy gene has not been located.     

The residue 129 polymorphism in human prion protein does not confer susceptibility to Creutzfeldt-Jakob disease by altering the structure or global stability of PrPC

There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129. This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains. Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP(C)). Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP(C). Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP(C); rather, its influence is likely to be downstream in the disease mechanism. We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP(Sc)) or intermediates or on the kinetics of their formation.