Immuno-gold staining for electron microscopy:

Gold nano-particles coated with protein A are used to detect antibody-bound protein by

transmission electron microscopy. (a) First antibodies are allowed to interact with their

specific antigen (eg, DHFR) in a section of fixed tissue. Then the section is treated with

electron-dense gold particles coated with protein A from the bacterium S. aureus (protein

A is a bacterial protein that binds very tightly to the Fc domains of antibody molecules).

Binding of the bound protein A to the Fc domain of the previously added antibodies (in

this case, anti- DHFR antibodies) makes the location of the target protein (DHFR) visible

in the electron microscope.

Fig 9-21 (Lodish, 6th ed.) (9-29, 7

th ed.)

3(c)

BIO 3302 JG Burr X-linked Adrenoleukodystrophy (ALD) and the movie, “Lorenzo’s Oil” ALD is a rare X-linked disorder characterized by the accumulation of elevated levels of certain very long chain saturated fatty acids (vlcFA) (C24:0 lignoceric acid and C26:0 hexacosanoic acid) in the membranes of several cell types, especially in the Schwann cells that create the myelin sheath around axons. The accumulation of these lipids leads to neural demyelination (immune mediated?). The most devastating type of ALD is the childhood cerebral form. About 40% of ALD cases are of this type, in which symptoms usually appear between 4 and 8 years of age. These boys become totally disabled within 6 months to 2 years after the onset of symptoms, and usually die sometime thereafter. ALD is a peroxisomal disorder, caused by a mutation in the ABCD1 gene (also known as the “ALD” gene.) The ABCD1 gene encodes an “ABC” type of pump that transports very long chain lipids (in their activated CoA-ester form) across the peroxisomal membrane, where they can then be oxidized by peroxisomal enzymes. The amount of vlcFA in cells is determined by the balance between biosynthesis and dietary intake, on the one hand, versus (on the other hand) oxidation in peroxisomes. Because of the defective ABCD1 transporter, vlcFAs are not oxidized in peroxisomes and accumulate in cell membranes. In the early 1980s, Augusto and Michaela Odone learned that their young son Lorenzo had the cerebral form of ALD. Augusto, an Italian banker, began to research the disease, and learned that oleic acid (C18:1) inhibits the enzyme responsible for the biosynthesis of vlcFAs. He began supplementing his son’s diet with glycerol trioleate oil. He deduced later that that another fatty acid, erucic acid (C22:1), would be also be a potent inhibitor of the biosynthesis of vlcFAs, so he added glycerol trieuricate to the glycerol trioleate he was giving to Lorenzo. He found that these oils significantly reduced the level of the toxic vlcFAs in his son’s blood. This story was made into a 1992 movie called “Lorenzo’s Oil”. To this day, a 4:1 combination of glycerol trioleate and glycerol trieuricate is referred to by physicians as Lorenzo’s oil. In controlled clinical trials, it has been confirmed that Lorenzo’s oil, in combination with dietary restriction of long chain fatty acids, is effective in normalizing serum levels of the C24 and C26 vlcFAs in ALD children. Unfortunately, dietary restriction of long chain fatty acids, and the administration of Lorenzo’s oil, might at best only slow the onset of disease symptoms, and then only if administered very early; these treatments are unable to actually reverse the symptoms of ALD. The movie had a happy ending, but in fact the real story of Lorenzo, was not such a happy one. Lorenzo survived into adulthood, but was paralyzed and able to communicate only with eye blinks and finger movements. He died of pneumonia at age thirty in 2008. The beginnings of a real happy ending might be provided by the recent discovery that the drug 4-phenyl-butyrate (4BPA) (“Buphenyl”) promotes the expression of a peroxisomal protein (“ALDRP”) functionally and structurally related to the ALD gene product. Treatment of tissue culture fibroblasts from ALD patients with 4BPA partially restores vlcFA oxidation in peroxisomes! (News & Views article by R.A. Wanders, Nature Medicine 4 (11), 1245-1246 [November, 1998]: “A happier sequel to ‘Lorenzo’s Oil?’”) Buphenyl treatment, in combination with dietary restriction and Lorenzo’s oil, might well be an effective treatment for ALD. At the very least, it might buy some children the time they need until effective gene therapy becomes available.

Lorenzo Odone 4 (b)

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