Pig Liver Tissue Research Articles

The amphipathic peptide mellitin as a tool to study the membrane-dependent activation of tissue transglutaminase

The role of membrane phospholipids on the cross-linking activity of guinea pig liver (tissue) transglutaminase has been investigated using the amphipathic model peptide melittin as glutaminyl substrate and the primary amine monodansylcadaverine as extrinsic amine donor. A marked increase of transglutaminase catalytic activity was observedin vitro assays in the presence of neutral membrane phospholipids. In contrast, activation was abolished in the presence of membranes containing pure anionic lipids. Enzyme activation could be ascribed to a direct binding of the lipid to the protein as demonstrated in enzymatic assays using a non membrane-interacting peptide (Cbz-Gln-Gly). The data obtained with model peptides suggest that the cross-linking activity of tissue transglutaminase could be modulated by the local microenvironment composition of the lipid bilayer and indicate that membrane phospholipids should be taken into account for further experiments directed to asess, the still poor understood, physiological role of tissue transglutaminase.

Lysine-Rich Histone (H1) Is a Lysyl Substrate of Tissue Transglutaminase: Possible Involvement of Transglutaminase in the Formation of Nuclear Aggregates in (CAG)n/Qn Expansion Diseases

Histone H1, which contains about 27% lysine, is an excellent lysyl donor substrate of Ca²⁺-activated guinea pig liver tissue transglutaminase as judged by rapid fluorescence enhancement in the presence of the glutaminyl-donor substrate 1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N-(5′N′N′-dimethylaminonaphthalenesulfonyl) diamidopentane. Sodium dodecyl sulfate gel electrophoresis of a 30-min reaction mixture revealed the presence of fluorescent high-M_r aggregates, which are also formed when histone H1 is incubated solely with activated tissue transglutaminase. Aggregate formation is even more pronounced when histone H1 is incubated with activated tissue transglutaminase and dimethylcasein (glutaminyl donor only). The findings suggest not only that histone H1 is an especially good lysyl substrate of tissue transglutaminase, but that it is also a glutaminyl substrate. Histone H1 is a good lysyl substrate of transglutaminase purified from Streptoverticillium mobaraense, suggesting that the ability of histone H1 to act as a transglutaminase lysyl substrate is widespread. In agreement with previous studies, it was found that human β-endorphin is a moderately good substrate of tissue transglutaminase. At least 8 neurodegenerative diseases, including Huntington’s disease, are caused by (CAG)_n expansions in the genome and by an expansion of the corresponding polyglutamine domain within the expressed, mutated protein. Polyglutamine domains are excellent substrates of liver and brain transglutaminases. A hallmark of many of the (CAG)_n/polyglutamine expansion diseases is the presence of polyglutamine-containing aggregates within the cytosol and nuclei of affected neurons. Transglutaminase activity occurs in both of these compartments in human brain. In future studies, it will be important to determine whether transglutaminases play a role in (1) cross-linking of histone H1 to glutaminyl donors (including polyglutamine domains) in nuclear chromatin, (2) the formation of nuclear aggregates in (CAG)_n/polyglutamine expansion diseases, (3) DNA laddering and cell death in neurodegenerative diseases and (4) depletion of neuropeptides in vulnerable regions of Huntington’s disease brain.

Cross-linking of Wild-type and Mutant α2-Antiplasmins to Fibrin by Activated Factor XIII and by a Tissue Transglutaminase

Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21) > Gln(419) > Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.

The induction of specific pig skin graft tolerance by grafting with neonatal pig thymus in thymectomized mice.

Xenogeneic donor-specific tolerance can be induced by transplanting fetal pig thymus and liver tissue (FP THY/LIV) to thymectomized (ATX), T/NK cell-depleted mice. By using neonatal pig tissue, we hoped to overcome two obstacles that arise with the use of fetal pig tissue: (1) the inability to keep fetal pigs alive after harvesting their thymic tissue, resulting in unavailability of their skin or other organs for grafting; and (2) the limited fetal thymic tissue yield, making application to large animals and humans more difficult. Neonatal pig thymus tissue (NP THY) was grafted into ATX, T/NK cell-depleted, 3Gy whole body-irradiated, originally immunocompetent B6 mice to evaluate the ability of NP THY to reconstitute mouse CD4+ T cells and to induce xenogeneic tolerance to donor pig skin grafts. Repopulation of mouse CD4+ T cells in the peripheral tissues was observed in T/NK cell-depleted, ATX B6 mice that received NP THY with or without neonatal pig spleen (NP SPL), but not in those receiving NP SPL alone, indicating that pig thymus grafting was necessary and sufficient for mouse T cell recovery. Seven of nine NP THY/SPL-grafted ATX mice and two of six NP THY-grafted ATX mice that reconstituted >5% CD4+ cells in PBL accepted donor pig skin long-term without lymphocyte infiltration, whereas they rejected allogeneic BALB/c skin and third party pig skin grafts as rapidly as euthymic mice. NP THY can support the development of mouse CD4+ T cells that are functional and specifically tolerant to donor pig antigens in ATX, T/NK cell-depleted, 3 Gy whole body-irradiated, originally immunocompetent B6 mice. Additional grafting of NP SPL with NP THY improves the efficiency of tolerance induction in this model.

Effect of dietary formates on growth performance, carcass traits, sensory quality, intestinal microflora, and stomach alterations in growing-finishing pigs.

Three experiments were conducted to evaluate the effect of adding salts of formic acid to diets for growing-finishing pigs. In Exp. 1, 72 pigs (23.1 kg and 104.5 kg initial and final BW) were used to evaluate the effect of Ca/Na-formate and K-diformate on performance and carcass traits. Treatments were organized in a 2 x 3 factorial arrangement with two feeding regimens (limit and semi-ad libitum feeding) and three diets (control, .85% Ca/Na-formate, and .8% K-diformate). No significant feeding regime x diet interaction was found. The K-diformate diet increased overall ADG of pigs compared with the control and Ca/Na-formate diets, but had no effect on ADFI or gain/feed (G/F) ratio. Neither K-diformate nor Ca/Na-formate had any effect on carcass lean or fat content. In Exp. 2, 10 limit-fed pigs (24.3 kg and 85.1 kg initial and final BW) were used to study the effect of K-diformate on performance and sensory quality of pork. Adding .8% K-diformate to diets increased ADG (P < .13) and G/F (P < .04), but had no effect on sensory quality of the pork or content of formate in liver, kidney, or muscle tissue of pigs. In Exp. 3, 96 limit-fed pigs (27.1 kg and 105 kg initial and final BW) were used to determine the effect of adding K-diformate to diets on performance, carcass traits, and stomach keratinization and(or) lesions. Adding K-diformate (0, .6, or 1.2%) to diets increased ADG and ADFI (linear P < .01). The K-diformate reduced the percentage of carcass fat (linear P < .03) and fat area in the cutlet (linear P < .09) and increased percentage lean in the ham (linear P < .01), flank (linear P < .02), loin (linear P < .09), and neck and shoulder (linear P < .09). The K-diformate had no negative effect on stomach alterations. In Exp. 3, the concentration of coliform bacteria in the gastrointestinal tract was evaluated in eight control and eight 1.2% K-diformate-fed pigs. The K-diformate reduced the number of coliforms in the duodenum (P < .03), jejunum (P < .02), and rectum (P < .10) of pigs. In conclusion, K-diformate improved growth performance and carcass quality of growing-finishing pigs, whereas Ca/Na-formate had no effect. K-diformate had no adverse effect on sensory quality of pork or on stomach alteration scores.

Simultaneous determination of seven penicillins in muscle, liver and kidney tissues from cattle and pigs by a multiresidue high-performance liquid chromatographic method

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction (SPE) was developed for determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in muscle, liver and kidney tissues of pigs and cattle. The compounds were extracted in aqueous solution by precipitation of organic materials with a mixture of sulphuric acid and sodium tungstate. The extract was cleaned up by SPE on a divinylbenzene-co- N-vinylpyrrolidone polymeric sorbent. Further clean-up was performed by liquid–liquid partition with diethyl ether. The extract was derivatised with benzoic anhydride and 1,2,4-triazole mercury (II) reagent. Chromatography was performed by reversed-phase gradient HPLC on a C 18 column with ultraviolet detection at 323 nm. The limits of detection estimated by a conservative model were in the range 8.9–11.1 μg/kg for amoxicillin, penicillin G, ampicillin, oxacillin, cloxacillin and nafcillin and 18.3–20.9 μg/kg for dicloxacillin. The mean recovery range was 66–77% for amoxicillin, 73–75% for penicillin G, 81–82% for ampicillin, 73–76% for oxacillin, 74–75% for cloxacillin, 66–72% for nafcillin and 58–65% for dicloxacillin.

Characterization of Glutathione S-Transferase of Taenia solium

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Addressing substrate glutamine requirements for tissue transglutaminase using substance P analogues

We have investigated the effect on the substrate requirements for guinea pig liver (tissue) transglutaminase of a set of 11 synthetic glutamine substitution analogues making up the full sequence of the naturally occurring tissue transglutaminase substrate substance P. While a number of peptide sequences derived from proteins that are well-recognized as tissue transglutaminase substrates have been studied, the enzyme activity using substitution analogues of full-length natural substrates has not been investigated as thoroughly. Thus, our set of substance P analogues only differs from one to other by one amino acid mutation while the length (of the peptide) is maintained as in the natural parent peptide. Our results indicate that a glutamine residue is not recognized as substrate by the enzyme whether it is placed at the N- or C-terminal or between two positively charged residues or between two proline residues. To further address the effect on enzyme activity of charged amino acids in the vicinity of the reactive glutamine residue, a new set of synthetic charge replacement analogues of substance P has been also studied. Together, the results have identified new minimal requirements for modification of a particular glutamine residue in a polypeptide chain. It would be of interest to set up a full set of such requirements in order to highlight potential glutamine residues as enzyme targets in the growing list of proteins that are being described as transglutaminase substrates.

Clinical and experimental study on regional administration of phosphorus 32 glass microspheres in treating hepatic carcinoma.

AIM:To study the therapeutical effectiveness, dosage range and toxic adverse effects of domestic phosphorus 32 glass microsphere and evaluate its clinical significance.METHODS:I.Fifty two BALB/c tumor bearing male nude mice were allocated into treatment group(n = 38) and control group(n = 14). In the former group different doses of (32) P-GMS were injected into the tumor mass, while in the latter (31)P-GMS or no treatment was given. The experimental animals were sacrificed in batches, and then the tumors and their nearby tissues were examined by light and electron microscopy.II. Through selective catheterization of hepatic artery, (32)P-GMS was infused to 5 healthy domestic pigs in a dosage equivalent to the therapeutic dose for human being, and (31)P-GMS was infused to another 5 healthy domestic pigs. Two pigs infused with contrast medium served as whole course blank controls. One pig from each group was surrendered to euthanasia at week 1, 4, 8 and 16 respectively. The ultrastructural histopath ological changes in liver tissues taken from different sites were evaluated semiquan titatively. III. One hundred and twenty seven times of (32)P-GMS intrahepatic artery interventional therapies were performed on 93 patients with hepatic carcinoma, including 79 cases of primary hepatic carcinoma and 14 cases of secondary hepatic carcinoma. (32)P-GMS (n = 30), and group B,(32)P-GMS and half dose of trans hepatic artery embolization (TAE)(n = 49), and 18 patients with HCC by TAE only as control group C. Fourteen patients with secondary hepatic carcinoma were treated in the same way as group B or C.RESULTS:I.Comparing with the control group, the treatment group of tumor bearing nude mice attained the tumor inhibition rates of 59.7%-93.7% (F = 579.62 P < 0.01) at 14d. At an absorbed dose of 7320Gy, the tumor cells were completely destroyed. When the absorbed doses ranged from 1830Gy to 3660Gy, most of the tumor cells showed the evidences of injury or necrosis, but there appeared some well differentiated tumor cells and enhanced effect of the autoimmunocytes. At an absorbed dose of 366Gy or less, some tumor cells still remained active prolix-ferative ability. The definite anticancer effect appeared as early as 3d after intratumoral injection of (32)P-GMS.II. The cumulative amount of (32) P-GMS in the target tissue after trans hepatic artery instillation attained more than 90% of the total dose administrated. Semiquantitative analysis of ultrastructral morphology in the experimental group showed no statistical difference between the nuclear abnormality (N(abn)) and mitochondrial variability (M(var)) at week 1 or 2, but revealed prominent difference (X(2) = 6.70-9.68, P < 0.01, X(2) = 65.09-115.09, P <0.001) as compared with those in the other groups. In the experimental group the N abn in tissues showed no significant difference between week 8 and week 16. No apparent changes were found in the stomach, spleen, kidney and lung tissues of the experimental pigs. III. The therapeutical results of HCC patients in group A were closely approximated to those of group C, no hematological toxic side effects were noted, and the systemic reaction was mild. In some patients 2mos-3mos after treatment some secondary foci appeared around the periphery of the primary lesion. In general better effectiveness was obtained in patients with small lesion. After analyzing by RIDIT method, the therapeutic result in group B was significantly better than that in group C, and secondary foci around the original lesion were rarely seen at 3mos after treatment. In group C the collateral circulation was reestablished along the periphery of primary foci and the secondary foci appeared more frequently, and were required to undergo several courses of treatment. In group B, 4 cases of HCC were treated surgically as their mass decreased in size after (32)P-GMS treatment.Resected specimens showed that the tumor was encapsulated by fibrotic tissue and most of the tumor cells necrosed. The 3 year survival rates were 43.3%-51.0% after A and B regimen treatment. In 14 cases of secondary HCC, the foci were well controled within one year after treatment.CONCLUSION:When the experimental model of implanted human liver cancer cells received (32)P-GMS of 1830Gy-3660Gy, it produced excellent anticancer effect without any injury to the normal neighboring tissues and the prominent anticancer effect was shown within 3d after intratumoral injection. Intrahepatic arterial administration of (32)P-GMS at the macro-cosmic absorbed dosage less than 190 Gy/dose exerted reversible sub lethal injury to domestic pig liver tissues. It took more than 8 weeks to repair the injured liver tissue and restore its function.(32)P-GMS trans hepatic artery embolization is an effective and safe regimen in treating hepatic carcinoma.

Effects of chromium picolinate on in vitro lipogenesis and lipolysis in adipose tissue and protein synthesis in liver tissue of pigs

Effects of chromium picolinate on in vitro lipogenesis and lipolysis in adipose tissue and protein synthesis in liver tissue of pigs

Pig MHC Mediates Positive Selection of Mouse CD4+ T Cells with a Mouse MHC-Restricted TCR in Pig Thymus Grafts

Remarkably normal immune function and specific T cell tolerance to discordant xenogeneic donors can be achieved by grafting fetal pig thymus and liver (FP THY/LIV) tissue to T cell and NK cell-depleted, thymectomized (ATX) mice. To determine whether or not host class II MHC molecules participate in the positive selection of mouse CD4+ T cells in FP THY/LIV grafts, we compared their development in ATX "AND" TCR-transgenic mice with positive selecting or nonselecting host MHC genotypes. Mouse TCR-transgenic CD4 single positive T cells repopulated the periphery significantly and to a similar extent in both T/NK cell-depleted, ATX AND mice with positive-selecting or nonselecting MHC backgrounds after grafting with FP THY/LIV. Therefore, MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing a host class II MHC-restricted TCR without a contribution from the host MHC. These results, in combination with previous studies performed in this model, suggest that the T cell repertoire that is generated by the combination of positive selection on xenogeneic MHC and negative selection on both recipient and xenogeneic porcine MHC is tolerant of both donor and recipient and has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of immunocompetence. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation and also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection.

ROLE OF REGULATORY CELLS DEVELOPING IN PORCINE THYMUS GRAFTS IN TOLERANCE TO BOTH HOST AND DONOR ANTIGENS

39 Specific tolerance to xenogeneic porcine donors and immune reconstitution can be achieved by grafting fetal pig thymic and liver tissue (FP THY/LIV) to T/NK cell-depleted, thymectomized (ATX) immunocompetent mice. Efficient intrathymic deletion of T cells with Vβ families which react with host superantigens or with a transgenic TCR that recognizes host MHC antigens is observed in FP THY grafts. Thus, negative selection of mouse thymocytes occurs upon recognition of host antigens in FP THY grafts. However, some of these mice (<5%) eventually develop an autoimmune-like disease, as indicated by clinical signs, such as weight loss, GVHD-like changes in the skin, and pathological examination. After adoptive transfer of CD4+ cells from these ill-appearing FP THY-grafted ATX B6 mice with B6 bone marrow cells to secondary lethally irradiated syngeneic B6 mice, the secondary recipients developed similar clinical signs, demonstrating that CD4+ cells play a key role in this phenomenon. Furthermore, adding naive syngeneic splenocytes to these inocula reduced the severity of illness in secondary recipients, suggesting that the absence of suppressor cells, which are present in normal mice, may be a cause of the autoimmune syndrome. The late failure of host-specific tolerance in a small fraction of mice correlates with a loss of graft function, suggesting an intrathymic origin for cells that suppress autoimmunity. On the other hand, mouse CD4+ cells from healthy FP THY/LIV-grafted BALB/c mice had the ability to suppress anti-pig responses of naive T cells. BALB/c nude mice that received naive BALB/c splenocytes i.p. rejected pig skin grafts with median survival time (MST) of 22.5 days. The co-transfer, with naive spleen cells, of tolerant splenocytes from FP THY/LIV-grafted BALB/c nude mice prolonged donor pig skin graft survival to a MST of 32.5 days (p<0.01). These studies suggest that mouse regulatory cells developing in discordant xenogeneic FP THY grafts play a role in the tolerance to donor antigens, and possibly to host antigens.

Tissue Transglutaminase-Catalyzed Formation of High-Molecular-Weight Aggregatesin VitroIs Favored with Long Polyglutamine Domains: A Possible Mechanism Contributing to CAG-Triplet Diseases

To investigate possible biochemical mechanisms underlying the “toxic gain of function” associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-Mraggregates—a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral–pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptidesin vitro,resulting in the formation of high-Mraggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-Mrprotein aggregates, thereby contributing to cell death.

Design and in vitro results of a high intensity ultrasound interstitial applicator

Interstitial technologies were proposed for the treatment of deep-seated and unresectable tumours. This study was designed to demonstrate the possibility of producing coagulation necrosis for a short exposure duration (20 s) and a large volume with an interstitial ultrasonic applicator equipped with a plane transducer. The applicator was evaluated in terms of electroacoustic efficiency (58% at 10.7 MHz), temperature pattern and in vitro pig liver tissues destruction. The temperature elevation reached 52, 39, 22 and 15 °C at, respectively, 2.5, 5, 7.5 and 10 mm from the applicator surface along the propagation axis. The evident cigar-shaped lesions spread on a depth of (8 ± 2) mm from the surface applicator, (10 ± 1) mm high and (3 ± 1) mm wide. The temperature measurements showed the limitations encountered with a 10 MHz transducer. The high increase in temperature of tissues close to the transducer can induce vaporisation and a gaseous barrier opaque to ultrasound. Owing to the shape and the operating frequency of the transducer, we hope to obtain the same satisfactory performances in vivo with weakly perfusion-dependent lesions. Axial and longitudinal applicator motions will enable us to treat more important volumes.

Design and preliminary results of an ultrasound applicator for interstitial thermal coagulation

The extracorporeal high-intensity focused ultrasound (HIFU) techniques are not still transposable for tumors of the digestive tract because of their locations. This study was designed to evaluate the feasibility of interstitial applicators (3.8 mm O.D.) to comply with this therapeutic lack and to demonstrate the possibility of producing coagulation necrosis by a specially designed probe and with a short exposure time (20 s). The active surface of the applicators consists of plane water-cooled PZT transducers working at 10 or 5 MHz. They were evaluated in terms of acoustic power emitted as a function of the frequency, and applied electrical input (electroacoustic efficiency of 75% at their working frequency) and in vitro and in vivo pig liver tissue destruction. The in vitro and in vivo necroses depth from the applicator surface ranged from 8 to 20 mm. This showed the advantage of a nondivergent source: the pressure decay is only due to the tissue absorption in the Fresnel zone. The lesions dimensions are slightly dependent on perfusion: 8 ± 2 mm deep in vitro for a 10.7-MHz transducer working at 14 W/cm2 against 10 mm in vivo. Operating at 5 MHz makes it possible to increase the therapeutic heating depth. For example, at a similar close-to-transducer temperature, the 5-MHz applicator induced, at a depth of 10 mm, a temperature elevation of 27°C against 15°C for that working at 10 MHz.

Stability of Sulfonamide Antibiotics in Spiked Pig Liver Tissue During Frozen Storage

A bulk portion of homogenized pig liver tissue was spiked at room temperature with 0.2 mg/kg (twice the Australian maximum residue limit) of each of sulfathiazole, sulfachlorpyridazine, sulfadimidine (sulfamethazine), sulfaquinoxaline, and sulfadimethoxine. After subsampling and packaging, selected individual packaged units were tested to confirm homogeneity of the prepared material. The material was stored frozen at -20 degrees C and analyzed in replicate by liquid chromatography on 11 sampling dates over a period of about 6 months. Analytical data were plotted on a log-linear scale and subjected to linear regression on the basis of first-order kinetics for the decay. Storage stabilities (decay half-lives at -20 degrees C) calculated from the mean slope of regression lines were sulfadimethoxine, 567 days; sulfadimidine, 457 days; sulfachlorpyridazine, 312 days; sulfathiazole, 291 days; and sulfaquinoxaline, 271 days. Significant depletion (65% loss) of residue was observed for sulfaquinoxaline during preparation of spiked bulk liver tissue. An extension of the study to measure the storage stability of sulfaquinoxaline under accelerated decay conditions (refrigerator temperature, 4 degrees C) showed it to be relatively unstable, with a decay half-life of 11 days. Results demonstrate the need for both regulatory agencies and testing laboratories to be aware of potential errors associated with improper transport, storage, and handling of tissue samples submitted for antibiotic testing.

Positive and negative selection of functional mouse CD4 cells by porcine MHC in pig thymus grafts.

Specific tolerance to discordant xenogeneic donors can be achieved by grafting of fetal pig thymic and liver tissue (FP THY/LIV) to T cell and NK cell-depleted, thymectomized (ATX) mice. Mouse CD4+ T cells develop in FP THY/LIV grafts, and demonstrate remarkably normal immune function, including host-restricted responses to keyhole limpet hemocyanin. We have therefore studied the role of host MHC class II in the development of mouse T cells in FP THY/LIV grafts by comparing their development in ATX MHC class II-deficient (IIKO) and wild-type (H-2b) mice. Mouse CD4+ T cells repopulated T/NK cell-depleted, ATX IIKO mice after grafting with FP THY/LIV, indicating that pig MHC can positively select mouse CD4 cells. Expression of TCR, MHC class I, Qa-2, heat-stable Ag, and CD45RB among double-positive and CD4 single-positive (SP) graft thymocytes in wild-type recipients was similar to that in normal mouse thymi, whereas CD4 SP thymocytes in grafts of IIKO mice showed increased Qa-2 and decreased heat-stable Ag expression, suggesting an increased level of maturity. Double-positive cells in grafts of IIKO mice also expressed higher than normal levels of Qa-2. Deletion within the grafts of Vbeta3+, Vbeta5.1/5.2+, and Vbeta11+ but not Vbeta6+, Vbeta7+, or Vbeta8.1/8.2+ mouse CD4 SP thymocytes in ATX IIKO mice demonstrated that swine leukocyte Ag participates in negative selection of the T cell repertoire. Therefore, porcine MHC mediates positive and negative selection of mouse thymocytes, but host class II MHC molecules also regulate thymocyte maturation in xenogeneic thymic grafts.

Polyglutamine domains are substrates of tissue transglutaminase: does transglutaminase play a role in expanded CAG/poly-Q neurodegenerative diseases?

Huntington's disease and six other neurodegenerative diseases are associated with abnormal gene products containing expanded polyglutamine (poly-Q; Qn) domains (n > or = 40). In the present work, we show that glutathione S-transferase (GST) fusion proteins containing a small, physiological-length poly-Q domain (GSTQ10) or a large, pathological-length poly-Q domain (GSTQ62) are excellent substrates of guinea pig liver (tissue) transglutaminase and that both GSTQ10 and GSTQ62 are activators of tissue transglutaminase-catalyzed hydroxaminolysis of N-alpha-carbobenzoxyglutaminylglycine. The present findings have implications for understanding the pathophysiological mechanisms of expanded CAG/poly-Q domain diseases.

Immune restoration by fetal pig thymus grafts in T cell-depleted, thymectomized mice.

Donor-specific tolerance can be induced across a discordant xenogeneic barrier in T/NK cell-depleted, thymectomized (ATX) B10 mice by grafting of fetal pig thymic and liver tissue (FP THY/LIV) under the kidney capsule. We have now examined the phenotype and function of murine T cells that develop in FP THY/LIV grafts in these mice. Mouse CD4+ T cells reached normal levels in PBL by 14 wk, and were maintained up to 30 wk. Similar proportions of splenic CD4+ cells expressed the naive phenotype (CD45RBhighMEL-14+CD44low) in FP THY/LIV graft recipients and euthymic control mice. These CD4 cells were functional, demonstrating normal proliferative responses and up-regulation of CD25 and CD69 after activation by mitogens or alloantigens. They proliferated in response to the protein Ag KLH presented by host MHC following in vivo immunization. ATX B10 mice grafted with FP THY/LIV also cleared Pneumocystis carinii infections, whereas simultaneously-treated ATX B10 mice not receiving FP THY were unable to do so. Discordant xenogeneic thymus grafting can therefore restore immune competence. Thus, in addition to tolerance induction, xenogeneic thymic replacement might have a potential role in the reconstitution of immunity in patients afflicted with immunodeficiencies affecting the thymus.

Localization of transglutaminase in hippocampal neurons: implications for Alzheimer's disease.

The purpose of this investigation was to identify and localize tissue transglutaminase (TGase) within neurons from the hippocampi of normal aged individuals and of those with confirmed Alzheimer's disease (AD). This enzyme may be a factor in the molecular mechanisms of neurodegeneration and formation of insoluble macromolecular complexes found in the neurons of normal aged and AD brain tissue. An antibody made to the extracellular TGase, coagulation factor XIIIa, was found to be specific for purified intracellular guinea pig liver tissue TGase. The specificity for liver tissue TGase has enabled us to identify tissue TGase(s) within rat hippocampal neurons and within neurons from normal aged and AD hippocampal tissues. Degenerating neurons from the AD hippocampus, compared to neurons from the normal aged hippocampus, exhibited increased immunoreactivity for TGase and demonstrated co-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associated with the neurofibrillary degeneration observed in AD, thereby implicating TGase as a potential factor in the pathogenesis of Alzheimer's disease.