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T Huff

Publications and source records attributed to T Huff.

At least 19 recordsLinked to original sources
PubMed2005

Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease.

BACKGROUND AND AIMS: Intestinal inflammation in coeliac disease is driven by the gluten fraction of wheat proteins. Deamidation or cross linking of gluten peptides by tissue transglutaminase (tTG), the coeliac disease autoantigen, creates potent T cell stimulatory peptides. Therefore, our aim was to identify the reaction patterns of gluten peptides, intestinal extracellular matrix proteins, and tTG. METHODS: tTG activity was analysed by incorporation of monodansyl cadaverine into gliadins. Fluorescence labelled tTG reactive short gliadin peptides were used to demonstrate their deamidation and explore their cross linking patterns with tTG itself or extracellular matrix proteins. Patient sera and controls were checked for autoantibodies to matrix proteins. RESULTS: Gliadins alpha1-alpha11, gamma1-gamma6, omega1-omega3, and omega5 were substrates for tTG. tTG catalysed the cross linking of gliadin peptides with interstitial collagen types I, III, and VI. Coeliac patients showed increased antibody titres against the collagens I, III, V, and VI. CONCLUSIONS: tTG formed high molecular weight complexes with all tested gliadins. As all tested gliadins were substrates for tTG, the tTG catalysed modifications were not restricted to single gliadin types and epitopes. Furthermore, haptenisation and long term immobilisation of gliadin peptides by tTG catalysed binding to abundant extracellular matrix proteins could be instrumental in the perpetuation of intestinal inflammation and some associated autoimmune diseases in coeliac disease.

Animals↗
PubMed2002

Chemotherapeutic drugs change actin skeleton organization and the expression of beta-thymosins in human breast cancer cells.

PURPOSE: Elevated expression of the beta-thymosin isotypes T beta(4), T beta(10), and T(15) appears to be involved in the manifestation of a malignant phenotype of human tumor cells, including those of mammary carcinomas. This has evoked an interest in these peptides as diagnostic/prognostic tumor markers. If increased levels of beta-thymosins correspond to tumor malignancy, the question arises whether tumor growth inhibition induced by chemotherapeutic drugs would reduce their expression. METHODS: Two human breast cancer cell lines, the estrogen receptor(ER)-positive MCF-7 and the ER-negative MDA-MB231, were thus analyzed for the amount of beta-thymosin mRNAs by RNase protection assay and for the respective peptide levels by HPLC following different hormonal and drug treatments. RESULTS: Both cell lines, growing in medium with 10% FCS, contain T beta(4) (400-500 fg/cell) and Tbeta(10) (about 100 fg/cell), but no T beta(15). Incubating MCF-7 cells with tamoxifen (1 microM) for 5 days resulted in about 80% growth inhibition and in reduction of intracellular T beta(4) and T beta(10) concentrations by about 40%. Levels of T beta(4) and T beta(10)-mRNA were reduced by about 60%. In contrast, cisplatin (2 microM) changed neither the peptide concentrations nor the mRNA levels of beta-thymosins, in spite of marked growth inhibition. In addition, no changes in beta-thymosin expression were observed in MDA-MB231 cells treated with either drug. MCF-7 cells maintained in estrogen-poor medium (10% horse serum) or stimulated to grow with estradiol (1 nM) had Tbeta(4) and T beta(10) concentrations reduced by about 30%, but changes in T beta(4)- and T beta(10)-mRNA levels did not correspond to those of the peptide. CONCLUSION: Expression of T beta(4) and T beta(10) mRNAs and their peptides is differentially regulated and does not correlate with growth. Instead, reduced beta-thymosin expression may be linked to more intensive TRITC-phalloidin staining of F-actin lining the membrane at sites of intimate cell-cell contacts, while increased beta-thymosin levels appear in cells with more extensive substrate adhesion. This suggests that beta-thymosins play a role in cell surface dynamics.

Actins↗
PubMed2001

beta-Thymosins, small acidic peptides with multiple functions.

The beta-thymosins are a family of highly conserved polar 5 kDa peptides originally thought to be thymic hormones. About 10 years ago, thymosin beta(4) as well as other members of this ubiquitous peptide family were identified as the main intracellular G-actin sequestering peptides, being present in high concentrations in almost every cell. beta-Thymosins bind monomeric actin in a 1:1 complex and act as actin buffers, preventing polymerization into actin filaments but supplying a pool of actin monomers when the cell needs filaments. Changes in the expression of beta-thymosins appear to be related to the differentiation of cells. Increased expression of beta-thymosins or even the synthesis of a beta-thymosin normally not expressed might promote metastasis possibly by increasing mobility of the cells. Thymosin beta(4) is detected outside of cells in blood plasma or in wound fluid. Several biological effects are attributed to thymosin beta(4), oxidized thymosin beta(4), or to the fragment, acSDKP, possibly generated from thymosin beta(4). Among the effects are induction of metallo-proteinases, chemotaxis, angiogenesis and inhibition of inflammation as well as the inhibition of bone marrow stem cell proliferation. However, nothing is known about the molecular mechanisms mediating the effects attributed to extracellular beta-thymosins.

Actins↗
PubMed2000

Androstenetriol and androstenediol. Protection against lethal radiation and restoration of immunity after radiation injury.

Androstenetriol (AET) and Androstenediol (AED) upregulate host immunity, leading to increased resistance against infections. AET augments IL-2, IL-3, IFN gamma levels, and counteracts hydrocortisone immune suppression. AET and AED at a dose of 0.75 mg/- and 8.0 mg/25-g mouse, protected 60 and 70%, respectively, of C57/BL/6J mice irradiated with a lethal dose. These hormones also protected mice irradiated with 6 Gy and infected with a coxsackievirus B4 LD50. AET significantly increased spleen lymphocyte numbers at 7, 14, and 21 days after a 6-Gy exposure. Fluorescent activated cell-sorter analysis of irradiated mice, spleen, and bone marrow showed that AET significantly augmented the myeloid precursor markers, CD11b/Mac-1, and B220 (pan B), as well as the absolute numbers of CD4+/CD8+ cells over the 21 days of testing. Overall, the data are consistent with AET/AED inducing a more rapid recovery of all hematopoietic precursors from the small number of surviving stem cells.

Anabolic Agents↗
PubMed1999

Thymosin beta(4) serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin.

Thymosin beta(4) possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin beta(4) serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin beta(4) may serve as a useful tool for further investigations in cell biology. Thymosin beta(4) could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin beta(4) concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin beta(4) may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.

Actins↗
PubMed1998

The dipyridyls paraquat and diquat attenuate the interaction of G-actin with thymosin beta4.

Beta-thymosins sequester G-actin and preserve a pool of monomers of actin which constitute an important prerequisite for cellular function of the microfilament system. To study the influence of paraquat binding to G-actin on the interaction of G-actin with thymosin beta4 we determined the apparent dissociation constant of the G-actin-thymosin beta4 complex in the absence or presence of paraquat using an ultrafiltration assay. Paraquat (1,1'-dimethyl-4,4'-dipyridylium dichloride) attenuates this interaction in a concentration- and time-dependent manner. When exposed to 10 mM paraquat, the apparent dissociation constant increased 10-85-fold within 15 min to 24 h. After incubation for 24 h even a paraquat concentration as low as 100 microM increased the dissociation constant of the G-actin-thymosin beta4 complex from 0.66 microM to 0.82 microM (P < 0.05). Diquat (1,1'-ethylene-2,2'-dipyridylium dibromide) similarly weakens the interaction of G-actin and beta-thymosins. In none of the experiments was oxidation of the methionine residue or any other modification of thymosin beta4 detected. Therefore we conclude that the dipyridyls paraquat and diquat directly interact with G-actin and thereby impede the interaction between G-actin and thymosin beta4.

Actin Cytoskeleton↗
PubMed1998

Plant profilin induces actin polymerization from actin : beta-thymosin complexes and competes directly with beta-thymosins and with negative co-operativity with DNase I for binding to actin.

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.

Actins↗
PubMed1997

Mapping the binding site of thymosin beta4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin.

The beta-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin beta4 was determined by analysing the binding of thymosin beta4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin beta4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin beta4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin beta4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin beta4 was obtained only when using the chemically cross-linked actin-thymosin beta4 complex, indicating that thymosin beta4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin beta4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

Actins↗
PubMed1997

C-terminal truncation of thymosin beta10 by an intracellular protease and its influence on the interaction with G-actin studied by ultrafiltration.

Two beta-thymosins are expressed in most mammalian tissues. We detected small amounts of a third peptide in extracts of rabbit spleen. The portion of this peptide increased when the tissue was first frozen and then thawed at 4 degrees C. Small amounts of the peptide are also present in cells from suspension cultures homogenized immediately in diluted perchloric acid. By means of amino acid analysis and MALDI-mass spectroscopy this peptide was identified to be a C-terminally truncated form of thymosin beta10. Having studied the formation in more detail we found that after a 4-h thaw at 4 degrees C all thymosin beta10 was truncated to thymosin beta10(1-41), which was further degraded during the next 20 h. On the other hand, thymosin beta4Ala, the second beta-thymosin being present in rabbit spleen, was not truncated or degraded even after 22 h. It might be possible that in vivo a truncated form of thymosin beta10 is formed by a carboxydipeptidase while thymosin beta4Ala is rather stable against proteolytic modification. By using a newly designed ultrafiltration assay, we determined the dissociation constants of the complexes of G-actin and these three beta-thymosins to be 0.28, 0.72, and 0.94 microM for thymosin beta4Ala, beta10, and thymosin beta10(1-41), respectively. The complex with beta4Ala is unambiguously more stable than the complex with beta10 or beta4 (0.81 microM). The change in the dissociation constant generated by the truncation of the two C-terminal amino acid residues of beta10 is small but statistically significant. This demonstrates that even the very last amino acid residues at the C-terminus of beta-thymosins are involved in the interaction with G-actin.

Actins↗
PubMed1997

Peer discussion on training physicians to be competent communicators: support for a multiple discourse approach.

To consider medical educators' views about training physicians to be competent communicators, a focus group was conducted at a large southeastern medical college with faculty responsible for training interns and residents to be competent communicators. The medical school was undergoing a curriculum review and instructional development process, including an examination of the methods being used to teach communication. A discussion of a teaching faculty committee revealed several themes, including the foundational assumption that communication is a natural ability that some have and others do not. Other notions likely to affect the educators' beliefs about the importance and effectiveness of communication education include the ideas that courtesy is synonymous with good communication and that although a few fundamentals might be transmitted, experience is the best teacher. Participants identified several audiences, in addition to patients, with whom they communicate as part of their profession, including other physicians, other health care providers (e.g., nurses and lab technicians), and hospital administrators, health care insurers, and others responsible for evaluating physicians' performance. The implications of this project for the development of medical communication courses within a multiple discourse approach are discussed.

Communication↗
PubMed1996

The expression of stem cell factor and its receptor, c-kit in human endometrium and placental tissues during pregnancy.

Stem cell factor (SCF) and its receptor Kit regulate the proliferation and survival of early hematopoietic cell types as well as germ cells and melanocytes. As SCF augments the effects of several hematopoietic growth factors that are produced in reproductive tissues during pregnancy and also plays an important role in cell migration, proliferation, and survival, we studied the expression and localization of this receptor/ligand in human endometrial and placental tissues. Kit was detected by Western blot analysis in early decidual and placental tissues (8-19 weeks gestation) and in term placenta. Immunohistochemistry localized Kit mainly in trophoblast and to a lesser extent in scattered cells in the placental villous core and decidual stroma. Ribonuclease protection assay showed that SCF messenger ribonucleic acid (mRNA) expression increased 3-fold in decidua from early pregnancy compared to proliferative and secretory endometrium (P < 0.05). Placental tissues expressed 4- to 8-fold higher levels of SCF mRNA compared to decidus (P < 0.05). Isolated placental villous core expressed 7-fold higher levels of SCF mRNA than did trophoblast (P < 0.05). Thus, SCF and its receptor Kit are expressed in human endometrium and placental tissues during pregnancy, and the pattern of receptor/ligand expression suggests that endometrial and placental villous core SCF may have a paracrine effect on trophoblast through the receptor Kit.

Base Sequence↗
PubMed1995

Interactions of beta-thymosins, thymosin beta 4-sulfoxide, and N-terminally truncated thymosin beta 4 with actin studied by equilibrium centrifugation, chemical cross-linking and viscometry.

All beta-thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and beta-thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several beta-thymosins, oxidized thymosin beta 4 and N-terminally truncated beta 4. The apparent dissociation constants for actin from bovine heart and beta-thymosins were 2.5, 0.1, and 2.7 microM for thymosin beta 4, [Ala1]beta 4(beta Ala4), and beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin beta 4 or beta Ala4. In rabbits thymosin beta Ala4 replaces beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin beta 4 at Met6 (beta 4-sulfoxide) as well as truncation of 6 [beta 4-(7-43)] or 12 [beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin beta 4-sulfoxide or beta 4-(7-43) or beta 4-(13-43), only beta 4-sulfoxide and not the truncated beta-thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-beta 4 complex is necessary to observe a comparable dissociation constant.

Actins↗
PubMed1993

c-myc expression correlates with suppression of c-kit protooncogene expression in small cell lung cancer cell lines.

The mRNAs encoding the c-kit protooncogene tyrosine kinase receptor and its ligand, hemopoietic stem cell factor, are coexpressed in the majority of small cell lung cancer cell lines, suggesting that an autocrine growth loop may exist. Functional c-kit protein levels correspond well with mRNA levels in these cells. We have observed that those cell lines which express the c-kit gene also express either the L- and N-myc genes; those cell lines which express the c-myc gene do not express the c-kit gene. We have determined, by analyzing several small lung cancer cell lines transfected with a c-myc expression vector, that heterologous expression of c-myc correlates with a marked down-regulation of c-kit expression. Regulation of c-kit expression by the myc gene family may be partly responsible for the differing biological properties of cell lines and tumors which express N- and L-myc versus those that express c-myc.

Carcinoma, Small Cell↗
PubMed1993

Localization of rat tryptase to a subset of the connective tissue type of mast cell.

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.

Animals↗
PubMed1992

Pseudomyxoma peritoneii: treatment with the argon beam coagulator.

The argon beam coagulator facilitates tumor debulking of peritoneal carcinomatosis. A patient with pseudomyxoma peritoneii originating from the left ovary with multiple peritoneal and omental implants was treated with the argon beam coagulator, enabling ablation of all gross disease.

Cystadenocarcinoma↗
PubMed1985

Lectin binding in the diabetic rat kidney.

In this study metal-conjugated concanavalin A (Con A) and Bandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of control rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper. Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB4 preincubation. BSA II binding also increased significantly with diabetes.

Animals↗
PubMed1984

Induction of human gamma interferon by structurally defined polypeptide fragments of group A streptococcal M protein.

The presence of interferon (IFN) has been demonstrated previously (i) in fluids obtained from the middle ears of children with Streptococcus pneumoniae infections, (ii) from the serum of mice injected intraperitoneally with either S. pneumoniae or Streptococcus pyogenes, and (iii) from human lymphoid cell cultures treated with a variety of bacteria. In this study, we showed that highly purified peptic extracts of three different serotypes of group A streptococcal M protein (pep M5, pep M6, and pep M24) stimulated human peripheral leukocytes to produce IFN. IFN production was apparent by 10 h and peaked 24 h after exposure. Dose-response experiments indicated that IFN could be detected in cultures treated with concentrations of M protein as low as 6 micrograms/ml, whereas maximum IFN production occurred at a concentration of 200 micrograms/ml. The IFN had antigenic and physicochemical characteristics of IFN-gamma. Preliminary leukocyte fractionation studies revealed that the IFN-producing cell was a nonadherent lymphocyte with receptors for sheep erythrocytes (T cell). Rabbit antisera specific for these structurally defined polypeptide fragments of streptococcal M protein (pep M5, pep M6, and pep M24) blocked IFN induction by each of the polypeptides. The data suggest that the different serotypes of streptococcal M protein may induce IFN by a common structural determinant shared by each of the polypeptide fragments tested.

Amnion↗