Detection of PrPsc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents.

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

The tobacco etch viral 5' leader and poly(A) tail are functionally synergistic regulators of translation.

The 5' cap (m7GpppN) and the poly(A) tail of eukaryotic mRNAs work in concert to establish an efficient level of translation in vivo. Nevertheless, several mRNAs naturally lack a cap or a poly(A) tail. Determining how these messages effectively compete for the translational machinery not only reveals alternative mechanisms for translational competence, but can also underscore similarities between alternative mechanisms and the standard cap/poly(A) tail interaction. The genomic RNA of tobacco etch virus (TEV), a potyvirus, is a polyadenylated mRNA that naturally lacks a cap (m7GpppN) at the 5'-terminus and yet is a highly competitive mRNA during translation. The 144-nt 5'-leader is largely responsible for directing efficient translation and can greatly increase the translational competence of reporter mRNAs. We have examined the synergy between the TEV 5'-leader and the poly(A) tail in transfected plant and animal cells. The TEV 5'-leader functioned optimally as a regulator of reporter mRNA translation only when a poly(A) tail was present. The effect of the TEV 5'-leader on the translation of capped transcripts was significantly less than that for uncapped mRNAs, suggesting that the TEV 5'-leader and the cap may promote similar steps in translation.

A phylogenetically conserved sequence within viral 3' untranslated RNA pseudoknots regulates translation.

Both the 68-base 5' leader (omega) and the 205-base 3' untranslated region (UTR) of tobacco mosaic virus (TMV) promote efficient translation. A 35-base region within omega is necessary and sufficient for the regulation. Within the 3' UTR, a 52-base region, composed of two RNA pseudoknots, is required for regulation. These pseudoknots are phylogenetically conserved among seven viruses from two different viral groups and one satellite virus. The pseudoknots contained significant conservation at the secondary and tertiary levels and at several positions at the primary sequence level. Mutational analysis of the sequences determined that the primary sequence in several conserved positions, particularly within the third pseudoknot, was essential for function. The higher-order structure of the pseudoknots was also required. Both the leader and the pseudoknot region were specifically recognized by, and competed for, the same proteins in extracts made from carrot cell suspension cells and wheat germ. Binding of the proteins is much stronger to omega than the pseudoknot region. Synergism was observed between the TMV 3' UTR and the cap and to a lesser extent between omega and the 3' UTR. The functional synergism and the protein binding data suggest that the cap, TMV 5' leader, and 3' UTR interact to establish an efficient level of translation.

Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques.

The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.

Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: new insights into the conformational changes that result in cAMP-dependent activation.

Previous studies of the ligand regulation of the cAMP-dependent protein kinase have demonstrated the cAMP-mediated dissociation of the holoenzyme by using nonequilibrium techniques; i.e., gel filtration, ion-exchange chromatography, and differential centrifugation. While physically mild, these could have caused weakly associated species to dissociate, thereby providing a potentially flawed interpretation of the mechanism of activation of the protein kinase. To assess this, the activation of the cAMP-dependent protein kinase has been monitored under equilibrium conditions using dipolar fluorescence energy transfer to measure changes in the proximity relations between the catalytic (C) and regulatory (R) subunits that compose the holoenzyme. Specifically, we prepared a heterochromatically labeled protein kinase type II holoenzyme, with the regulatory and catalytic subunits labeled with sulforhodamine and carboxyfluorescein, respectively, and monitored the exchange of electronic excitation energy between the C and R subunits by both donor lifetime and steady-state fluorescence. Biochemically, the heterochromatic holoenzyme was closely identical to the native protein with regard to cAMP-induced increase in catalytic activity, reassociation of C and R subunits, inhibition of catalytic activity by the specific protein kinase inhibitor (PKI), and observed dissociation examined by gel filtration upon cAMP addition. However, under equilibrium conditions, the energy-transfer measurements revealed that the addition of cAMP to this heterochromatic reporter complex promoted an estimated 10-A increase in the distance between the derivatization sites on C and R but not a dissociation of these subunits. Addition of PKI plus cAMP promoted full dissociation of the two subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity.

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K).

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.

Normal egg hatchability requires the simultaneous administration to the hen of 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol.

A previous report [Science 201, 835-837 (1978)] presented evidence that the combined and simultaneous administration of the cholecalciferol (D3) metabolites 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] and 24R,25-dihydroxycholecalciferol [24R,25(OH)2D3] to White Leghorn hens was necessary for embryo development and normal egg hatchability; in the absence of 24R,25(OH)2D3 none of the fertile eggs hatched. The present study extends this fundamental observation to a second species, the Japanese quail, Coturnix coturnix japonica and compares the biological actions of the two stereoisomers of the 24,25(OH)2 metabolite, namely the naturally occurring 24R,25(OH)2D3 and its unnatural epimer 24S,25(OH)2D3. Groups of 12-14 vitamin D-depleted adult female Japanese quail were mated with normal male quail and eight consecutive batches of eggs (25-41 eggs from each group) were placed in an egg incubator, and egg hatchability for the fertile eggs monitored on days 21 and 22. The egg hatchability (in percent +/- SD) for each group was: D3 (56.5% +/- 12.8); 1 alpha,25(OH)2D3 (1.3% +/- 2.5); 24R,25(OH)2D3 (29.6% +/- 3.1); 24R25(OH)2D3 + 1 alpha,25-(OH)2D3 (32.8%); and 24S,25(OH)2D3 + 1 alpha,25(OH)2D3 (7.2%). Also for all treatment groups the blood level of the expected vitamin D metabolites were in the normal range, and there were no significant differences in the embryo weights and eggshell thickness (of both hatched and unhatched eggs). These results indicate that the Japanese quail have the inherent capability to discriminate between the stereoisomers of 24,25(OH)2D3 and therefore strongly support the concept that only the naturally occurring 24R,25(OH)2D3 has an identifiable, unique biological role which is different from that of 1 alpha,25(OH)2D3.