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From: TSS ()
Date: August 10, 2006 at 1:03 pm PST


DID you all notice the Liquid Medicated recall for BSE ;



Red Cell, Iron Rich Homogenized, Yucca Flavored Vitamin-Iron-Mineral
Supplement for all classes of horses. For Animal Use Only. NET
of Farnam Companies, Inc. PO Box 34820, Phoenix AZ 85067-4820,
Recall # V-002-2.
Redglo, EQUICARE (brand), Homogenized Energy Building Liquid Multi-
Vitamin Supplement for Horses. EQUICARE PRODUCTS, A
Division of Farnam Companies, Inc., PO Box 34820, Phoenix, AZ,
Recall # V-003-2.
All codes.
Farnam Companies, Inc., Phoenix, Arizona, sent a recall letter dated
March 8, 2001, to all distributors via regular first class mail. Firm
initiated recall is ongoing.


The products contain protein material derived from
bovine mammalian tissues; however, the bags are not labeled with the
required BSE cautionary statement.


14,000 to 15,000 gallons.



ZuPreem Feline Diet, 14.0 oz cans for Non-Domesticated
Carnivores in the families Fedlidae, Canidea, and Hyenadea.
Recall # V-115-4.
6910 S1 SF01 military time of production: 06:05.
Menu Foods, Inc., Pennsauken, NJ, by telephone on February 2, 2004. FDA initiated recall is complete.
LACF Feiline Diet for non-domestic carnivores does not carry the BSE warning statement.
1,620 cases.

Product is a horse supplement packed into a 5 lb. Plastic
container with a yellow/green/red/brown label printed in
part "FORMULA: Each 5 pounds contains: Chondroitin Sulfate
27,000 mg D-Glucosamine HCL 100,000 mg Collagen hydrolyzed
27,000 MSM (methysulfonylmethane) 2,500 mg. Carti-Flex
COMPLEX net wt. 5 lbs, a concentrated natural joint
supplement containing Glucosamine HCL, Chondroitin sulfate,
Hydrolixed collagen and MSM". Recall # V-116-4.
All codes without the required cautionary statement are under recall.
Interfarma Corporation, Miami, FL, by letters on February 4, 2004. FDA initiated recall is ongoing.
This animal feed product does not contain the required BSE cautionary statement: "Do Not Feed to Cattle or Other Ruminants".
80 cases 4-5 lb. Containers per case.
Venezuela, Nicaragua and Guatemala.



Greetings again,

I thought I might bring this topic back up. The last time I voiced my concerns about this I was kicked off and banned from posting to the CVM_BSE-L@LIST.NIH.GOV Gov. list. Can you believe that! BUT I still am concerned, and would kindly like to ask, ounce for ounce and dose for dose, would there be a difference in dose rate by volume comparing the solid mad cow protein to the liquid mad cow protein, and what about ingestion and uptake via liquid compared to solid mad cow protein ???

History of TSS posting to CVM_BSE-L@LIST.NIH.GOV ,

They had allowed me to post from ;

-------- Original Message --------
Subject: BSE has been diagnosed in a cow born in December 1999 and February 1998
Date: Fri, 6 Aug 2004 11:46:53 -0500
From: "Terry S. Singeltary Sr."
Reply-To: "Terry S. Singeltary Sr."


-------- Original Message -------- Subject: Rejected posting to CVM_BSE-L@LIST.NIH.GOV
Date: Mon, 28 Feb 2005 10:51:27 -0500
From: "L-Soft list server at NIH (1.8e)"
To: "Terry S. Singeltary Sr."

You are not authorized to send mail to the CVM_BSE-L list from
your flounder@WT.NET account.


-------- Original Message -------- Subject: TSS BOOTED FROM CVMHomeP@CVM.FDA.GOV Date: Fri, 4 Feb 2005 13:00:11 -0600 From: "Terry S. Singeltary Sr." Reply-To: Bovine Spongiform Encephalopathy To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE ##################### Bovine Spongiform Encephalopathy ##################### Greetings list members,THIS is just for documentation only. i was on this list for a while,conversing, posting etc. (mainly to myself;-). I even hadto O.K. these messages like here on the BSE-L.PLEASE NOTE where they state;>>This has never been an active list serve,>THAT is totally false.I posted 49 messages from 8/6/04 to 12/23/04.having to ok them and then getting confirmations and such.THIS is one of MANY automated replies when i postedsomething. have many of these filed away;-------- Original Message --------Subject: Gail Schneitler/Marketing/SIS is out of the office.Date: Tue, 5 Oct 2004 16:01:23 -0400From: GSchneitler@usiinc.netTo: "Terry S. Singeltary Sr." I will be out of the office starting 10/05/2004 and will not return until10/07/2004.Will be in the Conyers office or at 336-577-4049.===============ONE of my final replies to a message/question;-------- Original Message --------Subject: Liquid StorageDate: Fri, 3 Dec 2004 11:37:11 -0600From: "Wagner, Garry W." Reply-To: "Wagner, Garry W." To: CVMUPDATE-L@LIST.NIH.GOVHi, Does anyone have rules concerning bulk storage of medicated liquid feeds at a retailers or dealers location, and if you do would you share them? Garry W WagnerPlant IndustriesNorth Dakota Department of Agriculture600 E Boulevard Ave Dept 602Bismarck North Dakota 58505-0020(701) MY reply;-------- Original Message --------Subject: Re: Liquid StorageDate: Fri, 03 Dec 2004 15:55:47 -0600From: "Terry S. Singeltary Sr." To: "Wagner, Garry W." CC: CVMUPDATE-L@LIST.NIH.GOVReferences: <>Hello Mr. Wagner,Wagner, Garry W. wrote:> Hi,>> Does anyone have rules concerning bulk storage of medicated liquid > feeds at a retailers or dealers location, and if you do would you > share them?>This may be of some use for you. IN fact, may be more than i shouldhave sent, but thought i might test the waters here to see how choppythey may be. I hope you find some use of this data...CVM Proposes Rules for Liquid Medicated FeedFDA is proposing to change the regu-lations for liquid medicated feed and free-choice medicated feed. By changing the regulations for liquid medicated feed, FDA intends to clarify: what data are required to demonstrate chemical and physical stability of a drug in liquid feed, how such data may be submitted for use in the new animal drug approval process, and which liquid medicated feeds may be manufactured in a feed manufacturing facility that has not obtained a medicated feed mill license from FDA. By changing the regulations for free-choice medicated feed, FDA wants to ensure that they are consistent with the requirements for liquid medicated feed, and that provisions for free-choice medicated feed and liquid medicated feed comply with the terms of the Animal Drug Availability Act (ADAA) of 1996 ( proposed rule was published in the May 28, 2003, Federal Register ( 98fr/03-12974.html ) Single copies of the proposed rule may be obtained by writing to the Communications Staff, FDA/Center for Veterinary Medicine, 7519 Standish Place, HFV-12, Rockville, MD 20855. Please send a self-addressed adhesive label to assist in processing your request.Submit written comments on the proposed rule to the Dockets Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Room 1061, Rockville, MD 20852. Submit electronic comments to FDA will consider all comments received by August 26, 2003. Comments on the proposed rule must be identified with Docket Number 93P-0174.Additional information is available in the May 28, 2003, Federal Register and from Dr. Dragan Momcilovic, Center for Veterinary Medicine (HFV-226), Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855, 301-827-0169, e-mail: dmomcilo@ .============================== few links to different states on this issue; Carolina Department of Agricultureand Consumer Services 2003> Because pet food> is exempt from the cautionary statement,> Do not feed to cattle or other ruminants,> inspections were performed that traced> salvaged pet food to the point of final> sale to ensure the product was properly> labeled and being fed to appropriate animals.> Under contract with FDA, a total of> 213 inspections were performed in 2003.> Of these, 189 inspections were for BSE> only and 24 were BSE and medicated feed> mill inspections. Following the Canadian> discovery in May of a cow with BSE, firms> using prohibited material were revisited> to determine compliance.> Inspections not under contract with> FDA are conducted at feed mills and firms> selling commercial animal and pet feeds> to determine if the firm is following good> manufacturing practices. A total of 4,417> inspections were conducted in 2003. During> these inspections, 3,205 product> samples were taken for analysis to determine> quality compliance. A total of 308> samples or 9.6 percent were out of compliance,> resulting in $29,842 in penalties.> Samples of feed intended for ruminant> animals were collected for quality compliance> and examined in the> lab for animal proteins. Annual Report(Covers January 1, 2002-December 31, 2002)Commercial FeedstuffsIdaho State Department of Agriculturesnip...> 2002 FEED VIOLATIONS SUMMARY> (543 registered feed companies  7600 registered commercial feed products)> (973 total samples of which 212 are violative  some samples have > multiple violations)> (38 stop-sales were issued for violative samples  some stop-sales > have multiple violations)> (4 stop-sales were issued administratively for label violations and/or > product contamination)snip... SUMMARY REPORTCommercial Feed InspectionsPLANT INDUSTRIES DIVISIONBUREAU OF FEED AND SEEDPETER W. HOFHERR, DIRECTORBureau of Feed and Seed2003 Feed Program Reviewsnip... these pets are fed back to ruminants for human/animal consumption.GOTTA love those FDA/USDA BSE/TSE triple firewalls.1: J Infect Dis 1980 Aug;142(2):205-8 Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to nonhuman primates. Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC. Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were exposed to the infectious agents only by their nonforced consumption of known infectious tissues. The asymptomatic incubation period in the one monkey exposed to the virus of kuru was 36 months; that in the two monkeys exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months, respectively; and that in the two monkeys exposed to the virus of scrapie was 25 and 32 months, respectively. Careful physical examination of the buccal cavities of all of the monkeys failed to reveal signs or oral lesions. One additional monkey similarly exposed to kuru has remained asymptomatic during the 39 months that it has been under observation.PMID: 6997404, Garry W. wrote:> Hi,>> Does anyone have rules concerning bulk storage of medicated liquid > feeds at a retailers or dealers location, and if you do would you > share them?>> Garry W Wagner>> Plant Industries>> North Dakota Department of Agriculture>> 600 E Boulevard Ave Dept 602>> Bismarck North Dakota 58505-0020>> (701) 328-1501>> >=====================THEN all of a sudden;-------- Original Message --------Subject: Re: greetings cvm_BSE-LDate: Wed, 22 Dec 2004 16:52:26 -0600From: "Terry S. Singeltary Sr." To: CVM HomePage References: <>Greetings CVM,>This has never been an active list serve, and it is not a discussion list.>well, it was active for a while when i was on it from8/6/04 to 11/10/04. I got about 40 messages through.I guess that last post did me in, can't blame you, it wasa barn burner for sure;> -------- Original Message --------> Subject: GENETIC MAKE-UP MAY DETERMINE WHAT TYPE OF CJD OCCURS WHEN > HUMANS ARE INFECTED WITH BSE> Date: Wed, 10 Nov 2004 14:34:35 -0600> From: "Terry S. Singeltary Sr." > Reply-To: "Terry S. Singeltary Sr." > To: CVM_BSE-L@LIST.NIH.GOV>>>>Ref: MRC/62/04>>Under strict embargo until 19.00 British Time Thursday 11 November 2004>>>GENETIC MAKE-UP MAY DETERMINE WHAT TYPE OF CJD OCCURS WHEN HUMANS ARE>INFECTED WITH BSE>>New research published today (19.00 hours Thursday 11th November) by a>team from the Medical Research Council (MRC) Prion Unit offers an>explanation about why only people with a particular genetic make-up have>so far developed vCJD. It also provides evidence that other types of>BSE-derived prion infection with a different pattern of symptoms might>occur in humans. The findings are published in the journal Science.>Just hope the little time i was allowed to post there, that the data i postedwas read. Anyway, thanks for letting me know and Merry Christmasto all at CVM...kindest regards,terryCVM HomePage wrote:>Dear Mr. Singeltary:>>This has never been an active list serve, and it is not a discussion list.>People other than authorized FDA/Center for Veterinary Medicine employees>are not authorized to post to it. >>Sincerely yours,>>FDA/CVM Home Page>>>-----Original Message----->From: Terry S. Singeltary Sr. [mailto:flounder@WT.NET] >Sent: Thursday, December 16, 2004 4:23 PM>To: CVM_BSE-L-request@LIST.NIH.GOV>Subject: greetings cvm_BSE-L>>Greetings CVM_BSE-L,>>I have been posting to the CVM_BSE-L@LIST.NIH.GOV since August of 2004 and>tried to post today and found that ;>>>-------- Original Message -------->Subject: Rejected posting to CVM_BSE-L@LIST.NIH.GOV>Date: Thu, 16 Dec 2004 16:04:34 -0500>From: "L-Soft list server at NIH (1.8e)" >To: "Terry S. Singeltary Sr." >>>>You are not authorized to send mail to the CVM_BSE-L list from>your flounder@WT.NET account. You might be authorized to send to the>list from another of your accounts, or perhaps when using another mail>program which generates slightly different addresses, but LISTSERV has no>way to associate this other account or address with yours. If you need>assistance or if you have any question regarding the policy of the>CVM_BSE-L list, please contact the list owners:>CVM_BSE-L-request@LIST.NIH.GOV.>>>>-------- Original Message -------->>Subject: Re: Protease-Resistant Human Prion Protein and Ferritin Are >Cotransported across Caco-2 Epithelial Cells: Implications for Species >Barrier in Prion Uptake from the Intestine>Date: Thu, 16 Dec 2004 15:06:58 -0600>From: "Terry S. Singeltary Sr." >To: CVM_BSE-L@LIST.NIH.GOV>>>======>>I was curious when I got the boot and for what reason?>>WILL I still be able to receive?>>thank you,>kind regards,>>Terry S. Singeltary Sr.>> >>>-------- Original Message -------->>Subject: Re: Protease-Resistant Human Prion Protein and Ferritin Are>>Cotransported across Caco-2 Epithelial Cells: Implications for Species >>Barrier in Prion Uptake from the Intestine>>Date: Thu, 16 Dec 2004 15:06:58 -0600>>From: "Terry S. Singeltary Sr">>To: CVM_BSE-L@LIST.NIH.GOV>>>>>>>>-------- Original Message -------->>Subject: Re: Protease-Resistant Human Prion Protein and Ferritin Are>>Cotransported across Caco-2 Epithelial Cells: Implications for Species >>Barrier in Prion Uptake from the Intestine>>Date: Wed, 15 Dec 2004 16:33:52 -0600>>From: "Terry S. Singeltary Sr." >>Reply-To: Bovine Spongiform Encephalopathy >>>>To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE>>References: <>>>>>>>>>##################### Bovine Spongiform Encephalopathy >>#####################>>>>Greetings list members,>>>>THE full text is to long and complicated to send through the list as >>html/text with all the bells and whistles attached, but thought i might >>post the discussion below for some that might want to comment on it. I >>would be most interested in comments on this study...>>>>kind regards,>>terry>>>>Discussion>>>>Here we provide insight into the pathway of PrPSc uptake and transport >>across intestinal epithelial cells. In particular, our data show that >>exposure of sCJD brain homogenate to DEs generates a C-terminal PrPSc >>core of 2730 kDa that is transported across Caco-2 cells in vesicular >>structures and that this process is not influenced by the level of >>endogenous PrPC expression. Within these vesicles, PrPSc is associated >>with ferritin, a major component of the PrPScprotein complex, and >>remains associated with ferritin after transcytosis. Because ferritin >>is normally absorbed from food and is abundantly present in a typical >>meat dish, these findings have important implications for prion uptake >> >>>>from contaminated food.> >>>Using the well tested in vitro model for evaluating intestinal>>uptake of selected food nutrients (Cereijido et al., 1978; Pinto et>>al., 1983; Glahn et al., 1998), we show the resilience of PrPSc to>>DEs and the facilitative effect of such treatment on PrPSc uptake>>by Caco-2 cell monolayers. We noted that after treatment of>>CJDH with stomach pepsin, PrPSc underwent limited proteolysis>>and comigrated with the C-terminal PKresistant>>core of PrPSc. Under similar conditions,>>PrPC in the NH was completely>>hydrolyzed. Much to our surprise, DEtreated>>PrPSc was transported across>>Caco-2 cells four times more efficiently>>than PK-treated PrPSc. We believe that>>this effect is attributable to the chaotropic>>effect of bile salts that disperse PrPSccontaining>>membrane phospholipds into>>small micelles, preventing the aggregation>>of PrPSc and facilitating its binding to epithelial>>cells. This observation has significant>>practical implications because there>>could be qualitative and/or quantitative>>differences in the digestive process between>>individuals and certainly between>>different species. Such differences, although>>subtle and apparently trivial, may>>influence host susceptibility to prion infection>> >>>>from contaminated food.> >>>Although purifying PrPSc from CJDH,>>we noted that theHand L chains of ferritin>>consistently cosediment with PrPSc. Resistance>>of the PrPScferritin complex to elution>>with low concentrations of salt and>>coimmunoprecipitation with either anti->>PrP or anti-ferritin antibodies suggested>>an association between the two proteins,>>rather than coincidental sedimentation.>>Remarkably, both the H and L chains of>>ferritin resisted PK and DE treatment and>>were associated with the protease-resistant>>core of PrPSc. Electron microscopic examination>>of the 8H4-immunoprecipitated>>material revealed fibrils decorated with>>ferritin aggregates. Although other proteins>>were detected by silver staining of>>8H4 and anti-ferritin immunoprecipitates>>attesting to the remarkably sticky nature of>>PrPSc, we believe that the association of>>PrPSc with ferritin is stronger and is more>>likely to be of biological significance. This>>notion is based on the fact that after repeated>>rounds of ultracentrifugation, only ferritin remained associated>>with PrPSc, and the complex could be dissociated only>>with 0.4 M NaCl. None of the other proteins copurified with>>PrPSc, suggesting that their coimmunoprecipitation with PrPSc is>>perhaps attributable to nonspecific interactions with the antibodies>>or with PrPSc itself (Morel et al., 2004). Whether the association>>of PrPSc and ferritin occurs in vivo or after homogenization>>of brain tissue is unclear from our data. Nevertheless, this complex>>is biologically significant because ingested PrPSc in contaminated>>meat undergoes a process similar to homogenization and>>DE treatment in the GI tract and is likely presented to the intestinal>>epithelium in a complex with ferritin. Interestingly, the>>-sheet-rich PrP peptide 106126 mixed with normal or CJD>>homogenate was not transcytosed effectively, indicating that the>>main determinant of PrPSc transport is not its -sheet-rich secondary>>structure. Preincubation of PrP106126, NH, or CJDH>>with exogenous purified ferritin did not facilitate the formation>>of coimmunoprecipitable PrPferritin complexes, indicating>>that the association of PrPSc with ferritin is more complex than a>>>>mere hydrophobic interaction during the>>process of homogenization. Regardless of>>the nature and site of PrPScferritin complex>>formation, this phenomenon is likely>>to influence the absorption of ingested>>PrPSc significantly, especially because ferritin>>in ingested food is known to undergo>>active absorption by the human intestinal>>epithelium (Murray-Kolb et al., 2003;>>Theil, 2003).>>Our results show that the PrPScferritin>>complex is endocytosed by Caco-2>>cells in vesicular structures that fuse to>>form phagosomes within the cell. Some of>>these vesicles are transcytosed intact to the>>BL chamber, much like the reported release>>of PrPSc-containing exosomes into>>the extracellular environment by epithelial>>cells (Fevrier et al., 2004). Sensitivity of the PrPScferritin >>transport to incubation at low temperature and treatment with>>brefeldin A and nocodazole suggest the involvement>>of an active transport process>>(Klausner et al., 1992). Although Caco-2>>cells are known to endocytose ferritin, the>>mechanistic details of this process remain>>elusive (Murray-Kolb et al., 2003). Specific>>receptors for ferritin have been reported>>on liver cells, lymphocytes, erythroblasts,>>oligodendrocytes, and on various cell lines>>(Mack et al., 1983, Harrison and Arosio,>>1996; Hulet et al., 2000). Our data demonstrating>>significant inhibition of PrPSc>>ferritin uptake in the presence of excess>>ferritin derived from human liver, spleen,>>or brain suggests the presence of a ferritinspecific>>receptor or a transporter on>>Caco-2 cells. The presence of such a receptor>>on epithelial cells and the close association>>of PrPSc and ferritin in digested food>>incriminate ferritin as a possible transporter>>of PrPSc across the intestinal epithelial>>cell barrier.>>>>Our data show that 3040% of ferritin from NH is consistently >>transcytosed across Caco-2 cells without degradation. In CJDH, this >>amount varies with the size of PrPSc-ferritin aggregates. Small, >>detergent soluble complexes are transcytosed intact, whereas large, >>detergent insoluble aggregates remain on the monolayer in the AP >>chamber (our unpublished observations). These large aggregates may be >>internalized via M-cells, FDCs, or dendritic cells as reported >>previously (Heppner et al., 2001; Huang et al., 2002). It is >>conceivable that endocytosed ferritin is packaged in distinct vesicles >>that are either targeted to lysosomes or transcytosed to the BL >>surface. The associated PrPSc in CJDH probably follows both routes, >>although the majority appears to be transcytosed because very little >>PrPSc was detected in cell lysates (our unpublished observations). This >>assumption is supported by the fact that a significant proportion of >>the PrPScferritin complex remains intact after transcytosis, as >>evidenced by coimmunostaining of endocytosed aggregates in M17 cells >>cultured in the BL chamber. PrPC from untreated NH did not show >>significant association with ferritin and was not transported to the BL>>chamber in several experiments. However, ferritin from untreated>>NH was detected consistently in the BL chamber (our>>unpublished observations). Thus, either PrPC is not endocytosed>>at all or is degraded within Caco-2 cells. A small amount of PrPSc>>was detected occasionally independent of associated ferritin. It is>>unclear whether this fraction is associated with another protein,>>is transported independently, or results from dissociation of the>>PrPScferritin complex in an intracellular compartment.>>The notion that PrPSc is cotransported with ferritin ignores>>the key requirements of host susceptibility to prion infection,>>such as the level of PrPC expression and the extent of homology>>between host PrPC and incoming PrPSc (Prusiner et al., 1990;>>Weissmann et al., 2002; Thackray et al., 2003). Although in apparent>>contradiction, our data suggest that the uptake of PrPSc>>and its subsequent replication are distinct processes. The former>>is independent of host PrPC, whereas the latter requires PrPC as>>substrate for additional replication. This hypothesis is supported>>by our data that show no influence of PrPC overexpression on>>PrPSc transport across Caco-2 cells and by a recent report demonstrating>>PrPC expression below the tight junctions of polarized>>epithelial cells, making it physically impossible for incoming>>PrPSc to come in contact with host PrPC (E. Morel et al., 2004a).>>>>The cotransport of PrPSc with ferritin raises important questions >>regarding prion uptake from contaminated food. Although this report >>uses a homologous experimental setup, ferritinHand L chains are known >>to share significant homology across species (Harrison and Arosio, >>1996) and may facilitate the transport of PrPSc from distant species >>across the intestine. Because PrPSc is notorious for its sticky nature, >>ferritin may be only one such carrier protein. The identification and >>functional role of other proteins associated with DE-treated PrPSc is >>important for fully understanding the mechanism of PrPSc uptake from >>ingested food and preventing a carrier state across species. >>Heterologous PrPSc in such carriers may be transported to sites where >>it may undergo conformational adaptation with time (Hill et al., >>2000; Race et al., 2001), or in the case of livestock, lie dormant >>until ingested by a susceptible host. Such apparently healthy >>carriers would disseminate PrPSc through a variety of means, posing a>>potential threat to the general population...END>>>>>>Figure 7. sCJDPrP Sc remains associated with ferritin after>>transcytosis. A, Caco-2 cell monolayers were placed in a 12-well>>culture dish containing M17 neuroblastoma cells cultured on coverslips >>in the BL chamber, and biotinylated CJDH-DE was added>>to the AP chamber (see Fig. 6 A). After an overnight incubation, Caco-2 >>cells on filters and M17 cells on coverslips were processed>>for immunostaining. Immunoreaction of filters with anti-ZO-1 shows the >>presence of tight junctions in all monolayers (green;>>panels 1, 5). Immunostaining of M17 cells in the BL chamber for PrP >>(green) and ferritin (red) shows colocalization of the two>>proteins, indicating the presence of intact PrP Scferritin complexes >>after transcytosis (panels2 4, arrows). Coimmunostaining of>>M17 cells for PrP (green) and streptavidin (red) (panels 68) confirms >>that the PrP signal is derived from biotinylated CJDH-DE in>>the AP chamber. Scale bar, 10 m. B, Electron microscopic analysis of >>the PrP Scferritin complex immunoprecipitated with 8H4>>shows fibrillar structures and membranes decorated with ferritin >>aggregates (top inset; arrows). When added to Caco-2 cells, the>>complex is internalized in relatively large phagosome-like structures >>surrounded by a single membrane (top; arrowheads). Some>>of these vesicular structures are extruded out from the BL surface of >>Caco-2 cells and are seen within the pore of the membrane>>filter (bottom; arrowhead). (The internalized material in phagosomes >>shows similar structures as observed in the immunoprecipitated>>material.) Scale bar: 0.25 m; inset,1.5. N, Nucleus; T, tight junction.>>AQ: I>>>>Figure 8. The binding of sCJDPrP Scferritin to Caco-2 cells is>>competitively inhibited by excess ferritin. A, Western blotting of>>NH Pellet and CJDH Pellet fractions with 3F4 reveals no reactivity with >>the NH sample but strong reactivity with N-terminally>>truncated PrP Sc bands from the CJDH Pellet sample (lanes 1, 2). >>Reblotting with anti-ferritin shows the presence of ferritin in both>>NH and CJDH samples (lanes 3, 4). Longer exposure reveals high molecular >>weight bands of PrP and ferritin that appear to>>comigrate (lanes 5 8, arrow). B, Caco-2 cell monolayers were incubated >>with biotin-tagged NH Pellet or CJDH Pelletbrain lipid>>mixture and processed for staining with Texas Redstreptavidin. Both NH >>Pellet and CJDH Pellet bind to the cell surface (panels 1, 2,>>Ferritin), and the binding is inhibited by preincubation of the cells >>with 1.5 g/ml human spleen ferritin (panels 3, 4,>>Ferritin).>>C, Caco-2 cells were incubated with 25 l of biotinylated CJDH-DE with >>no previous exposure to ferritin (Ferritin) or after>>preincubation with 1.5 g/ml human spleen ferritin (>>Ferritin) and processed for staining with Texas Redstreptavidin and>>8H4 anti-mouse FITC (panels 1 6). Mock-treated cells show PrP-specific >>immunoreactivity (green) that colocalizes with>>streptavidin (red) (panels 13). However, preincubation with ferritin >>abolishes PrP-specific staining significantly (panels 46).D,>>Caco-2 cells with no previous treatment (panel 1,NH Pellet) or after >>preincubation with NH Pelletbrain lipid mixture containing>>human brain-derived ferritin (panel 2,>>NH Pellet) were exposed to 25 l of CJDH-DE and immunostained with 8H4 >>anti-mouse>>FITC. Mock-treated cells show strong PrP-specific immunoreactivity >>(panel 1), which is lost significantly after pretreatment of the>>cells with NH Pellet (panel 2). Scale bar, 10 m.>>10 " J. Neurosci., January 5, 2005 " 25(5):?? Mishra et al. " Transport >>of Protease-Resistant PrP across Epithelial Cells>>balt6/zns-neusci/zns-neusci/zns-orig/zns9802-05a simmsl S4 11/16/04 >>13:18 Art: 1192112 Input-YY(v)>>AQ: J>>AQ: K>>TSS>>>>>>>>>>Terry S. Singeltary Sr. wrote:>>>> >>>>>##################### Bovine Spongiform Encephalopathy>>>#####################>>>>>>Neurobiology of Disease>>>Protease-Resistant Human Prion Protein and Ferritin Are Cotransported>>>across Caco-2 Epithelial Cells: Implications for Species Barrier in >>>Prion Uptake from the Intestine>>>>>>Ravi Shankar Mishra, * Subhabrata Basu, * Yaping Gu, Xiu Luo, >>>Wen-Quan>>>Zou, Richa Mishra, Ruliang Li, Shu G. Chen, Pierluigi Gambetti, >>>Hisashi Fujioka, and Neena Singh>>>>>>Institute of Pathology, Case Western Reserve University, Cleveland,>>>Ohio 44106>>>>>>Foodborne transmission of bovine spongiform encephalopathy (BSE) to>>>humans as variant Creutzfeldt-Jakob disease (CJD) has affected over >>>100 individuals, and probably millions of others have been exposed to >>>BSE-contaminated food substances. Despite these obvious public health >>>concerns, surprisingly little is known about the mechanism by which >>>PrP-scrapie (PrPSc), the most reliable surrogate marker of infection >>>in BSE-contaminated food, crosses the human intestinal epithelial cell >>>barrier. Here we show that digestive enzyme (DE) treatment of sporadic >>>CJD brain homogenate generates a C-terminal fragment similar to the >>>proteinase K-resistant PrPSc core of 27-30 kDa implicated in prion >>>disease transmission and pathogenesis. Notably, DE treatment results >>>in a PrPSc-protein complex that is avidly transcytosed in vesicular >>>structures across an in vitro model of the human intestinal epithelial >>>cell barrier, regardless of the amount of endogenous PrPC expression. >>>Unexpectedly, PrPSc is cotransported with ferritin, a prominent >>>component of the DE-treated PrPSc-protein complex. The transport of >>>PrPSc-ferritin is sensitive to low temperature, brefeldin-A, and >>>nocodazole treatment and is inhibited by excess free ferritin, >>>implicating a receptor- or transporter-mediated pathway. Because >>>ferritin shares considerable homology across species, these data >>>suggest that PrPSc-associated proteins, in particular ferritin, may >>>facilitate PrPSc uptake in the intestine from distant species, leading >>>to a carrier state in humans.>>>>>>Key words: prion infection; subclinical infection; PrP transport; new>>>variant CJD; ferritin; epithelial cell barrier; Caco-2>>>>>>--------------------------------------------------------------------->>>--->>>Received July 15, 2004; revised October 27, 2004; accepted November 2, >>>2004.>>>>>>>>>>>>TSS>>>>>>##############>>>##############>>>>>>>>> >>>>>############## >>##############>>######### ##########

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